drug discovery strategies methods

DRUG 

DISCOVERY 

STRATEGIES 

METHODS 

EDITED BY 

ALEXANDROS MAKRIYANNIS 

DIANE B~EGEL 

Center for Drug Discovery 

University of Connecticut 

Storrs, Connecticut, U.S.A. 

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

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Preface 

Drug research encompasses diverse branches of science united by a common 

goal, namely, developing novel therapeutic agents and understanding 

their molecular mechanisms of action. This process is a lengthy, exacting, 

and expensive undertaking that involves integration of data from different 

fields and culminates in the final product—a new drug in the marketplace. 

In the past decade, progress in drug research has flourished because of 

major contributions from a variety of disciplines. 

The material presented in this volume focuses on a number of 

research topics that have provided critical information in the field of drug 

discovery. Several chapters present techniques that extend our understanding 

of the three-dimensional structure of macromolecules, principally 

proteins, but also nucleic acid polymers and organized lipid and carbohydrate 

assemblies. As greater structural data on the these molecules become 

available, information can be obtained on their interactions with small 

endogenous ligand drug molecules as well as on the interactions between 

two or more of these biopolymers. Such knowledge enhances our overall 

understanding of the biochemical systems of interest and their relevance 

for therapeutic discovery. In addition to the basic knowledge gained by 

such research, the data provide a solid basis for the development of novel 

drugs with greater potencies, higher specificities of action, and reduced side 

effects. 

Another area of research covered in this book is the in vivo 

anatomical localization of potential therapeutics using PET and SPECT 

analysis (Chapter 5). These techniques allow researchers to pinpoint the 

localization of high-affinity ligands in the living organism with high 

accuracy, thus giving researchers a window on the functions of the brain 

and other organs and on the sites of action of potential therapeutic agents. 

Such studies will provide a blueprint for the design of pharmacological 


agents that will target specific regions of affected organs and deliver therapeutic 

actions rapidly and with high specificity. 

High throughput methods have increased our capacity for appropriate 

candidate compounds selection and also for developing libraries of 

novel compounds from which such candidates can be selected. Chapter 7 

discusses the use of solid-phase synthesis for the high throughput production 

of peptides and other small molecules. In addition, as discussed in 

Chapter 6 on peptidomimetics, the swift production of novel leads holds 

considerable promise for future discovery of novel therapeutic agents. 

The investigation of therapeutic targets for cannabinoid sites of action 

has already generated considerable interest within the field of drug 

discovery, and Chapter 4, which details the results of such studies, highlights 

the importance of target-based studies. The enhanced appreciation 

of the role of stereochemistry in drug action has focused efforts on understanding 

the conformation of drugs as they bind to their target receptor. 

Studies of the diverse effects of cannabinoids and the development of 

compounds that employ the information gleaned from the ligand/receptor 

data should provide substantial insight into their molecular mechanisms of 

action. Future research will promote the development of drugs that are 

capable of higher specificity. longer half-lives, and lessened toxicity. In 

studies of potential antiviral therapies, the understanding of viral target 

molecules is essential for the production of effective medications that interact 

specifically in the viral life cycle and gene products, which will result 

in lowering drug toxicity to the host and enhancing the antiviral activity of 

the pharmacotherapy. As the nature of viral infectivity, cell growth, death, 

and receptor biology are elucidated, the methods and paradigms for development 

of highly specific medications will provide superior treatments 

for a number of diseases that pose a terrible burden worldwide (Chapters 10 

and 11). 

From the fields of proteomics and genomics that hold significant 

promise for unique medications, several areas of biology have also found 

applications in the drug discovery arena. The study of regulatory molecules 

and oncogenes has opened new avenues in drug therapy, as discussed in 

Chapter 8 on G-protein-coupled receptors and Chapter 2 on SRC homology 

domains. Research on protein misfolding (Chapter 9), which has been 

implicated in neurodegenerative diseases, has highlighted the need to 

enhance our understanding of structural alterations in normal proteins 

products. Chapter 1 details the development of such research, and asserts 

that only as we understand the basic physical mechanisms of such alterations 

can new therapeutic regimens be proposed and tested. 


The topics included in this volume are not intended to be allinclusive. 

Our approach has been eclectic, in an effort to bring the reader 

the most exciting aspects of drug discovery, along with the methods that 

show the most promise in enhancing the discovery process. 

The chapters presented in this book have been contributed by specialists 

in their areas of research and will provide a contemporary picture of 

the overall field of drug discovery to scientists from diverse disciplines. 

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved. 

Alexandros Makriyannis 

Diane Biegel

Contents 

Preface 

Contributors 

1. Protein Crystallography in Structure-Based Drug Design 

Xiayang Qiu and Sherin S. Abdel-Meguid 

2. Src Homology-2 Domains and Structure-Based, Small- 

Molecule Library Approaches to Drug Discovery 

Chester A. Metcalf III and Tomi Sawyer 

3. Three-Dimensional Structure of the Inhibited 

Catalytic Domain of Human Stromelysin-1 by 

Heteronuclear NMR Spectroscopy 

Paul R. Gooley 

4. Cannabinergics: Old and New Possibilities 

Andreas Goutopoulos and Alexandros Makriyannis 

5. Development of PET and SPECT Radioligands for 

Cannabinoid Receptors 

S. John Gatley, Andrew N. Gifford, Yu-Shin Ding, 

Ruoxi Lan, Qian Liu, Nora D. Volkow, and 



6. Structural and Pharmacological Aspects of 

Peptidomimetics 

Peter W. Schiller, Grazyna Weltrowska, Ralf Schmidt, 

Thi M.-D. Nguyen, Irena Berezowska, Carole Lemieux, 

Ngoc Nga Chung, Katharine A. Carpenter, 

and Brian C. Wilkes 

7. Linkers and Resins for Solid-Phase Synthesis: 1997-1999 

Pan Li, Elaine K. Kolaczkowski, and Steven A. Kates 

8. Allosteric Modulation of G-Protein-Coupled 

Receptors: Implications for Drug Action 

Angeliki P. Kourounakis, Pieter van der Klein, 

and Ad P. I. IJzerman 

9. Protein Misfolding and Neurodegenerative Disease: 

Therapeutic Opportunities 

Harry LeVine III 

10. Uncoating and Adsorption Inhibitors of Rhinovirus 

Replication 

Guy D. Diana and Adi Treasurywala 

11. Profiles of Prototype Antiviral Agents Interfering 

with the Initial Stages of HIV Infection 

E. De Clercq 


Contributors 

Sherin S. Abdel-Meguid Suntory Pharmaceutical Research Laboratories, 

Cambridge, Massachussets, U.S.A. 

Irena Berezowska Clinical Research Institute of Montreal, Montreal, 

Quebec, Canada 

Katharine A. Carpenter Clinical Research Institute of Montreal, Montreal, 


Ngoc Nga Chung Clinical Research Institute of Montreal, Montreal, 


Eric De Clercq Rega Institute for Medical Research, Katholieke Universiteit 

Leuven, Leuven, Belgium 

Guy D. Diana ViroPharma, Inc. Exton, Pennsylvania, U.S.A. 

Yu-Shin Ding Brookhaven National Laboratory, Upton, New York, 

U.S.A. 

S. John Gatley Brookhaven National Laboratory, Upton, New York, 

U.S.A. 

Andrew N. Gifford Brookhaven National Laboratory, Upton, New 

York, U.S.A. 

Paul R. Gooley University of Melbourne, Parkville, Victoria, Australia 


Andreas Goutopoulos Serono Reproductive Biology Institute, Rockland, 

Massachusetts, U.S.A. 

Ad P. IJzerman Leiden University, Leiden, The Netherlands 

Steven A. Kates Surface Logix, Inc., Brighton, Massachusetts, U.S.A. 

Elaine K. Kolaczkowski Vertex Pharmaceuticals, Cambridge, Massachussetts, 

U.S.A. 

Angeliki P. Kouranakis University of Thessaloniki, Thessaloniki, 

Greece 

Ruoxi Lan University of Connecticut, Storrs, Connecticut, U.S.A. 

Carole Lemieux Clinical Research Institute of Montreal, Montreal, 


Harry LeVine III University of Kentucky, Lexington, Kentucky, U.S.A. 

Pan Li Vertex Pharmaceuticals, Cambridge, Massachusettes, U.S.A. 

Qian Liu University of Connecticut, Storrs, Connecticut, U.S.A. 

Alexandros Makriyannis University of Connecticut, Storrs, Connecticut, 

U.S.A. 

Chester A. Metcalf III ARIAD Pharmaceuticals, Inc., Cambridge, Massachusetts, 

U.S.A. 

Thi M.-D. Nguyen Clinical Research Institute of Montreal, Montreal, 


Xiayang Qiu SmithKline Beecham Pharmaceuticals, King of Prussia, 

Pennsylvania, U.S.A. 

Tomi Sawyer ARIAD Pharmaceuticals, Inc., Cambridge, Massachusetts, 

U.S.A. 


Peter W. Schiller Clinical Research Institute of Montreal, Montreal, 


Ralf Schmidt Clinical Research Institute of Montreal, Montreal, Quebec, 

Canada 

Adi Treasurywala Pfizer Central Research, Groton, Connecticut, U.S.A. 

Pieter van der Klein Leiden University, Leiden, The Netherlands 

Nora D. Volkow NIDA, Bethesda, Maryland, U.S.A. 

Grazyna Weltrowska Clinical Research Institute of Montreal, Montreal, 


Brian C. Wilkes Clinical Research Institute of Montreal, Montreal, 



1 

Protein Crystallography in 

Structure-Based Drug Design 

Xiayang Qiu 

SmithKline Beecham Pharmaceuticals, King of Prussia, 

Pennsylvania, U.S.A. 

Sherin S. Abdel-Meguid 

Suntory Pharmaceutical Research Laboratories, Cambridge, 

Massachusetts, U.S.A. 

I. INTRODUCTION 

Proteins are responsible for a wide variety of important biological functions 

in living organisms and are commonly used as targets of therapeutic 

agents. A unique primary and tertiary structure is a hallmark property of a 

protein. Although several related and even unrelated proteins may share 

the same overall tertiary structure or fold, each will differ from the others in 

the details. Knowledge of the detailed atomic three-dimensional structure 

of the protein and/or its ligand complexes should facilitate the design of 

novel, high affinity ligands that interact with that protein. The process of 

elucidating the atomic structure of proteins and their complexes, and the 

design of novel, therapeutically relevant ligands based on these structure 

elucidations, is known as structure-based drug design. 

Proteins are complex molecules, typically containing several thousand 

atoms. Although Pauling and Corey proposed the a helix and the h 

sheet as the main secondary structural elements of proteins in 1951, and the 

crystal structure of myoglobin was reported by John Kendrew in 1958, 


crystal structure determination in the early days were hampered by 

numerous technical limitations and usually required many years of hard 

work. By the mid-1980s, substantial improvements in data acquisition 

software and hardware had considerably accelerated the speed with which 

a crystal structure could be determined. Trying to capitalize on the 

potential of structure-based drug design, several pharmaceutical companies 

built their own protein crystallography laboratories, and a number of 

structure-based drug design efforts emerged in industrial and academic 

laboratories [1]. 

In the past 10 years, we have experienced a sudden burst in the 

number of protein three-dimensional structures determined. By the end of 

the twentieth century, merely 40 years since the first protein structure was 

solved, there were over 11,000 structures deposited in the Protein Data 

Bank (PDB). Although each entry is not a unique protein, the number of 

novel structures deposited in the PDB has increased sharply during the last 

decade. These proteins include not only soluble proteins, but also a number 

of membrane proteins. Furthermore, structures of protein–protein and 

protein–nucleic acid complexes, viruses, and the ribosome are also available. 

This marvelous scientific achievement was mostly credited to the 

method of single-crystal x-ray diffraction (protein crystallography), 

although a notable number of structures were determined by means of 

NMR spectroscopy. Many factors in addition to the incredible advances in 

computer hardware and software contributed to the improved efficiency 

and precision in protein crystallography: the advent of molecular biology, 

which allows for cloning, mutation, and overexpression of many targets 

that are difficult to isolate from natural sources; advances in protein 

purification that facilitate the production of large amounts of highly 

purified proteins; improvement in protein characterization and crystallization 

strategies; enhancement of data acquisition techniques and equipments; 

access to powerful synchrotron radiation sources; and introduction 

of the selenomethionine multiple-wavelength anomalous diffraction 

(MAD) procedure for phase determination. Currently, almost all large 

pharmaceutical and numerous biotechnology companies have established 

in-house macromolecular crystallography units, and the crystallographic 

community is solving thousands of new structures every year. With 

structural information becoming more readily available, structure-based 

drug design has become an integral part of the modern drug discovery 

process and has begun to contribute to a significant portion of the current 

drug discovery portfolio. 


Identifying and bringing a successful small-molecule drug to the 

market requires considerable effort, which typically costs millions of 

dollars and may span as much as10 years. With this time scale in mind, 

one must realize that structure-based drug design is still in its infancy, 

having started in earnest in the mid-1980s. While the concept of such a 

rational approach has been around for some time, for much of the work 

in the field it is still too early to demonstrate market successes. Moreover, 

although structural knowledge may be used for lead generation 

and lead optimization, or even for addressing some developability issues, 

it does little to address other important issues in drug development 

ranging from the appropriateness of targets or disease models to government 

regulatory issues or changing market forces. In fact, drug 

discovery is a risky business in that only a very small number of compounds 

are able to find their way to the market. Therefore, the successful 

structure-based design and the launch of inhibitors of HIV protease 

[2] and influenza virus neuraminidase [3] as drugs are particularly 

encouraging events for the field [4–9]. In this chapter, we will introduce 

the technique of protein crystallography and its use in structure-based 

drug design, point out the technical challenges ahead of us, and report 

many practical lessons learned during the past decade of structure-based 

drug design. 

II. THE DRUG DISCOVERY PROCESS 

The many steps of the complex and multidisciplinary drug discovery 

process can be grouped into four major phases: target identification and 

validation, lead identification, lead optimization, and biological testing 

(Fig. 1). Choosing an appropriate target is usually the first step in the drug 

discovery process. Target selection requires an understanding of human 

diseases and the biological processes that lead to a particular disease. 

Although historically drugs (e.g., h-lactams) were discovered without 

knowledge of their molecular target, knowing the target greatly enhances 

one’s ability to discover novel drugs in a timely fashion. Recent advances in 

sequencing the human genome, as well as the genomes of many human 

pathogens, have provided a large pool of potential novel molecular targets. 

Most future drug discovery efforts will start with a relatively unknown gene 

selected from a sequence database based on one or more attractive features 

that could provide a hint of its function, such as tissue distribution, genome 


Figure 1 Simplified drug discovery process. 

localization, and/or sequence homology or structural analogy to a known 

protein. Cloning, expression, purification, and characterization of the 

protein target and other tool reagents such as antibodies or receptors will 

usually follow, to be used in target validation with a set of appropriate 

genetic and biological assays. 

The second step is to identify a suitable lead molecule to interact 

with the molecular target. This is usually achieved through high throughput 

screening of available chemical compound libraries and natural 

products, typically containing hundreds of thousands of compounds. 

Although the size of the library per se is not critical, a library that contains 

a large number of molecules is essential to assure molecular diversity. 

Novel lead molecules can also be designed by analysis of the threedimensional 

structure of the target molecule in a process known as de 

novo design. A desirable lead should usually have at least low micromolar 

binding potency against the target and should be amenable to further 

synthetic manipulations. 

The third step is to optimize the lead molecule through iterative 

chemical synthesis and biological testing, aiming to obtain molecules with 

the required potency (typically nanomolar), selectivity, bioavailability, 

and DMPK (drug metabolism and pharmacokinetics) properties. This step 

usually requires considerable time and resources; usually the synthesis of 

hundreds of compounds is needed to deduce a robust SAR (structure– 

activity relationship). Such resources can be considerably reduced and the 


time significantly shortened if optimization employs knowledge from the 

three-dimensional structure of complexes of leads with the target. 

The last step of the drug discovery process involves the testing of lead 

compounds to address issues such as efficacy, bioavailability, and safety. 

Testing may include in vitro assays but ultimately would require a suitable 

disease model and studies in animals. Many compounds may need to be 

designed and synthesized to identify the one compound with all the desired 

properties. Such a compound can be advanced to preclinical studies and 

eventually to the clinic. 

III. THE STRUCTURE-BASED DRUG DESIGN CYCLE 

Timely optimization of lead compounds requires knowledge of the threedimensional 

structure of target–ligand complexes. Protein crystallography 

has been the predominant technique used to elucidate the three-dimensional 

structure of proteins in structure-based drug design. Crystallographic 

studies usually consume tens of milligrams of pure protein and 

take several months to yield the first crystal structure. Therefore, one 

should start crystallographic efforts as soon as suitable material is available, 

preferably even before initiation of high throughput screening. Once 

a lead has been identified through high throughput screening or de novo 

design, structure determinations of target–ligand complexes should be 

pursued. The use of information derived from the structure determination 

of the target bound to the initial lead molecule should allow for the design 

and synthesis of new ligands with improved properties, as well as the 

initiation of further rounds of structure-based design. Through iterations 

of structure determination, design, synthesis, and biological testing (Fig. 2) 

a drug candidate should emerge. 

In addition to lead optimization and lead identification, three-dimensional 

structures of the target–ligand complexes can contribute to the 

traditional drug discovery process in other ways. For example, structural 

information combined with genomic sequences may aid in target identification 

by helping to classify genes with unknown functions. Structures 

can be used as templates for de novo design or in silico lead identification 

through screening of virtual libraries. Structural information can provide a 

basis for the design of directed combinatorial libraries [10,11]. Moreover, 

structural studies of leads with serum albumin and various cytochrome 

P450s should allow for a better understanding of some of the developability 

issues that may arise during drug development. 


Figure 2 Structure-based design cycle. 

IV. PROTEIN CRYSTALLOGRAPHY 

For most noncrystallographers, protein crystallography tends be a black 

box full of jargon. Here, we give a brief overview of the technology in an 

attempt to demystify some of the terms used. 

A. Crystallization 

Obtaining large single crystals that diffract to high resolution remains the 

primary bottleneck of protein crystallography. The most widely used 


crystallization method is the hanging-drop method of vapor diffusion 

(Fig. 3), in which a drop (1 or 2 AL) of protein is mixed with an equal 

volume of a precipitant on a glass coverslip and is sealed over a well 

containing the same precipitant added to the protein. Many factors are 

known to be important in protein crystallization: protein purity (preferably 

>95% pure) and concentration (typically 10 mg/mL), the nature of 

precipitant [e.g., poly(ethylene glycol) or various salts] and its concentration, 

the nature, concentration, and pH of the buffer, the presence or 

absence of additives (e.g., metal ions, reducing agents, protease inhibitors, 

metal chelators, detergents) and effectors (e.g., ligands, cofactors, substrates, 

inhibitors), the rate of equilibrium between the protein and the 

precipitant, the crystallization temperature, and so on. Since there are no 

general rules to correlate all these factors to the eventual success in 

obtaining crystals, protein crystallization remains a trial-and-error process 

and a significant bottleneck in protein crystallography: failure rate is 

typically 50% even with thousands of crystallization trials. Many methods 

and techniques have been employed to enhance one’s ability to obtain 

protein crystals. Molecular biology and biochemical methods have been 

utilized to generate domains of large proteins that may be less flexible and 

thus more amenable to crystallization. Biophysical tools such as dynamic 

light scattering [12] and ultracentrifugation [13] have been used to study 

protein aggregation in solution. Molecular biology has been employed to 

generate mutants that do not aggregate or are more soluble. Crystallization 

trials using incomplete factorial designs [14] allow the screening of a 

much wider range of conditions with a modest number of experiments, and 

Figure 3 Protein crystallization: diagrammatic representation of the hangingdrop 

method of vapor diffusion. 


thus less protein. Miniaturization and automation made possible by the 

use of advanced crystallization robots may also have a great impact on the 

future success of protein crystallization. 

B. X-Ray Diffraction Data Acquisition 

The next step is to measure x-ray diffraction data from a single crystal 

(Fig. 4). Data are usually measured by means of an area detector 

such as a phosphorus image plate or a charge-coupled device (CCD). 

Through several steps of computational analysis, the position and 

amplitude or intensity of the each diffraction spot can be obtained. 

Because diffraction intensities are proportional to the volume of the 

crystal and generally decrease at higher resolution, protein crystals must 

be reasonably large to give strong enough diffraction signals at high 

resolution. While a cube of 0.1 to 0.5 mm in each dimension is still 

preferred by most crystallographers, the availability of powerful synchrotron 

radiation sources has made the analysis of much smaller 

crystals feasible. Crystals also must be stable enough in the x-ray beam 

to allow the measurement of a complete diffraction data set from a 

single crystal. In this regard, flash-freezing of protein crystals under 

proper conditions at cryogenic temperatures [15] has virtually eliminated 

radiation decay problems. 

Figure 4 Diagrammatic representation of single-crystal x-ray diffraction and 

data collection. 


C. Phasing 

The ultimate goal of an x-ray diffraction experiment is to produce an 

electron density map that is then used to build an atomic model of the 

molecule being studied (Fig. 5). The use of single-crystal x-ray diffraction 

techniques to determine the three-dimensional structure of molecules 

requires the measurement of amplitudes and the calculation of phases 

for each diffraction spot. Although amplitudes can be directly measured 

from diffracting crystals, as noted earlier, phases are indirectly determined. 

The inability to directly measure phases is known as the ‘‘phase 

problem’’ [16]. In practice, there are several ways to get around the phase 

problem. If the protein of interest is small (f100 amino acids) and highresolution 

data (1.2 A ˚ or better) are available, phases can be obtained 

computationally by using the so-called direct method. This is basically the 

same technique used to determine crystal structures of small organic 

molecules. If the protein being studied is known to have a fold similar 

to that of a protein with a known three-dimensional structure, one uses the 

molecular replacement (MR) method, in which the known structure serves 

as a model to generate approximate phases that are then refined against 

the experimental data obtained from crystals of the protein under study. 

Until recently, multiple isomorphous replacement (MIR) was the most 

widely used method for ab initio phase determination. This technique 

requires the introduction into the protein under study of atoms of high 

atomic number (heavy atoms) such as mercury, platinum, and uranium, 

without disrupting the protein’s three-dimensional structure or the 

packing in the crystal. This is achieved by soaking crystals in a solution 

Figure 5 Steps in the use of protein crystallography for structure determination. 


containing the desired heavy atom. The binding of one or more heavy 

atoms to the protein alters the diffraction of the crystals from that of 

the underivatized (native) crystals. If the introduction of heavy atoms is 

truly isomorphous, the differences between the diffraction of the 

derivative and of the native will represent only contributions from the 

heavy atom(s). Thus, the problem of structure determination is reduced 

to locating the position of one or a few heavy atoms. Once their 

positions have been accurately determined, the heavy atoms are used to 

calculate phases for all diffraction intensities. In theory, one needs only 

two isomorphous derivatives, but in practice more are needed owing to 

errors that are introduced in data measurement as well as the lack of 

isomorphism. Multiple-wavelength anomalous dispersion (MAD) phasing, 

cited earlier, has gained popularity in the last 10 years, and this 

more recent technique for ab initio phase determination is now the 

predominant method in de novo structure determination. In the MAD 

technique, cells that overexpress the protein can be grown in media 

containing selenomethionine (Se-Met) instead of methionine, producing 

proteins that have Se-Met at all the methionine positions. Because of 

the unique absorption quality of Se, diffraction data can be measured 

by using a Se-Met-substituted crystal at three or four different wavelengths 

around the Se absorption edge. These data can be analyzed by 

using computational methods to generate phase information, allowing 

an electron density map to be calculated [17]. Such an experiment calls 

for modern synchrotron facilities. 

D. Model Building and Refinement 

Once an electron density map has become available, atoms may be fitted 

into the map by means of computer graphics to give an initial structural 

model of the protein. The quality of the electron density map and 

structural model may be improved through iterative structural refinement 

but will ultimately be limited by the resolution of the diffraction 

data. At low resolution, electron density maps have very few detailed 

features (Fig. 6), and tracing the protein chain can be rather difficult 

without some knowledge of the protein structure. At better than 3.0 A ˚ 

resolution, amino acid side chains can be recognized with the help of 

protein sequence information, while at better than 2.5 A ˚ resolution 

solvent molecules can be observed and added to the structural model 

with some confidence. As the resolution improves to better than 2.0 A ˚ 

resolution, fitting of individual atoms may be possible, and most of the 


Figure 6 Electron density of an a-helix at different resolutions. 

amino acid side chains can be readily assigned even in the absence of 

sequence information. 

E. Understanding Structural Coordinates 

Once a crystal structure has been determined, the information is 

communicated in the form of an atomic coordinates file. In addition 

to a list of the atomic positions, the coordinates file contains other information 

that deserves an explanation and requires attention by the 

user. Some of the terms included in an atomic coordinates file are 

explained briefly. It is hoped that the information will provide the reader 

with insights to evaluate the quality of the structure, distinguish between 

its well-defined and flexible regions, and make sensible decisions in 

structural analysis. 

The unit cell is the basic microscopic building block of the crystal. A 

crystal can be viewed as a three-dimensional stack of identical unit cells, 

each defined by three cell edges (a, b, c, in angstroms), and three angles (a, 

h, g in degrees) between each pair of edges. Each unit cell may contain one 

or more protein molecules related by crystal symmetry. The unique portion 

of the unit cell (i.e., the portion that is not related to other portions by 

crystal symmetry) is called the asymmetric unit. There are only 230 different 

combinations of symmetry elements in crystals; each of these is called a 

space group. However, since biological molecules are enantiomorphic, 


which means that a protein crystal cannot contain mirror planes, the 

number of space groups of relevance to protein crystallography is reduced 

to 65. It is possible to have more than one copy of the same protein in an 

asymmetric unit. However, these will be related by ‘‘noncrystallographic’’ 

symmetry. Therefore, all atoms of an asymmetric unit, along with the unit 

cell dimensions and the space group, must be given in the coordinates file 

for subsequent analysis and for regenerating the structure in any portion of 

the unit cell or the crystal, which may be important for studying intermolecular 

‘‘crystal packing’’ interactions. 

The R-factor is probably the single most important number that 

provides a sense of the overall quality of the structure. It is defined as 

[A||Fobs| k*|Fcalc||] / A|Fobs|, where Fobs is the observed structure factor 

(the square root of the measured diffraction intensity or amplitude), Fcalc 

is the structure factor calculated from the model, and k is a scaling 

factor. The factor R is a measurement of the agreement between the 

structural model and the observed diffraction data; the lower the 

number, the better. For a refined crystal structure, the R factor is often 

approximately 10 times its resolution (e.g., 20% for a 2.0 A ˚ resolution 

structure). Along with the traditional R factor, most of the recent 

structures also report an Rfree value, which is obtained from the part 

of the diffraction data (5–10%) set aside and not used during structural 

refinement. Generally Rfree is 5–10% higher than R; larger discrepancies 

between the two may indicate that there is a problem in the structure 

model or diffraction data, or that the structure is overrefined against the 

data. Reducing R to below 20% used to be the goal for structural 

refinement; but obtaining a sensible Rfree is now considered to be more 

important. Therefore, before analyzing a crystal structure on computer 

graphics, one should check the R factor and Rfree values to get a sense of 

the overall quality of the structure. It is important to note that these 

values can be reported as percentages (20%) or as fractions (0.20). 

The atomic temperature factor, orB factor, measures the dynamic 

disorder caused by the temperature-dependent vibration of the atom, as 

well as the static disorder resulting from subtle structural differences in 

different unit cells throughout the crystal. For a B factor of 15 A ˚ 2 , displacement 

of an atom from its equilibrium position is approximately 0.44 

A ˚ , and it is as much as 0.87 A ˚ for a B factor of 60 A ˚ 2 . It is very important 

to inspect the B factors during any structural analysis: a B factor of less 

than 30 A ˚ 2 for a particular atom usually indicates confidence in its 

atomic position, but a B factor of higher than 60 A ˚ 2 likely indicates that 

the atom is disordered. 


For a particular crystal, the number of diffraction data increases as 

the resolution increases, which means that more experimental data will 

be available for structural refinement. There are four parameters to be 

refined for each atom: x, y, z (atomic position), and B (temperature 

factor). If the crystal has normal solvent content (i.e., about 50%), the 

number of experimental data and refinement parameters will be about the 

same at 2.8 A ˚ resolution. This suggests that B factors for individual 

atoms should be refined only when data have a resolution better than 2.8 

A ˚ . Refinement of atomic B factors at lower resolution will have no 

physical meaning, although a lower but meaningless R factor will result. 

Identification and refinement of solvent molecules (e.g., waters) become 

reliable only when the structure has at least a 2.5 A ˚ resolution. Even then, 

before a water molecule is used in mechanistic or computational analysis, 

it is always wise to check its B factor for the existence of at least one 

hydrogen bond to hold the water to the protein. At times, spurious water 

molecules are added (such additions will result in a meaningless lower R 

factor). Unless the structure has been determined at a reasonably high 

resolution, electron density and refinement often do not discriminate 

between the oxygen and nitrogen atoms of asparagines and glutamines, 

or the alternative conformations of histidine side chains. In a detailed 

structural analysis, it may be necessary to check alternative conformations 

of Asn, Gln, or His side chains and decide which one makes more 

sense chemically. 

V. IN SILICO LEAD GENERATION 

Armed with the crystal structure of the protein–ligand complex and upto-date 

computer modeling software, one can design additional ligands. 

Numerous molecular modeling software programs are available for that 

purpose. However, it is important to note that current computational 

algorithms have their limitations and utilize many approximations. Therefore, 

while computer modeling software has been proven useful [4,18], 

further testing and structural validations are required to identify the best 

possible compound. 

A. In Silico Screening of Virtual Compound Libraries 

Starting with the crystal structure of the target, it is possible to screen for 

leads in three-dimensional compound databases such as the Cambridge 


Structural Database [19] or the Chemical Abstracts Service Registry [20], 

or convert private databases to 3-D structures by programs such as 

CONCORD [21]. Several programs are available for such screening. For 

example, DOCK [22] works by using a set of overlapping spheres to create 

a complementary image of the ligand binding site and essentially matching 

the shape of a putative ligand with that of the image to generate a 

‘‘goodness of fit’’ score that is then used to rank the hits identified. Instead 

of comparing shapes, the program LUDI [23] uses parameters that 

describe hydrogen-bonding potential and hydrophobic complementarity 

to match the ligand and its binding site. These programs can rapidly search 

through three-dimensional databases of small molecules and rank each 

candidate. Typically, the 100 to 200 top-scoring compounds are examined 

graphically to identify the best 10 to 50 candidates for experimental testing. 

In the case of DOCK, 2 to 20% of these in silico hits may show micromolar 

binding affinity [4]. Subsequently, crystallography can be used to optimize 

any leads identified. 

B. Building Leads from Molecular Fragments 

Again starting with the crystal structure of the target, another strategy 

is to dock small chemical fragments into the ligand binding site, then 

grow the fragment to better complement the binding site. Programs 

such as GRID [24], AUTODOCK [25], and MCCS [26] can be used 

for the docking step. GRID uses small functional groups to probe the 

binding site and evaluate interaction energies by using an empirical 

Lennard-Jones energy function, as well as electrostatic and hydrogenbonding 

terms. AUTODOCK uses simulated annealing for ligand 

conformational search to dock small ligands of flexible conformations 

onto a rigid binding site and a standard force field for rapid grid-type 

energy evaluation. MCSS (multicopy simultaneous search) places thousands 

of copies of functional groups in the binding site and optimizes 

them simultaneously to generate energetically favorable positions and 

orientations in a flexible binding site. Once selected, suitable binding 

fragments can be built into a single compound by manual modeling or 

by using linking programs such as CAVEAT [27], which attempts to 

identify a suitable cyclic linker from a database. Alternatively, programs 

like GroupBuild [28] can search compound libraries for potential 

leads that have the functional fragments identified by the programs 

just described. 


VI. STRUCTURE-BASED LEAD OPTIMIZATION 

Once a chemical lead has been identified, the structure-based lead 

optimization process goes through several iterations of structure determination, 

design, chemical synthesis, and biological testing. The goal is 

to optimize the lead in terms of electrostatic interactions, van der Waals 

contacts, and the fit in the ligand binding pocket. The design process 

may be simple and intuitive if one starts with a relatively high affinity 

lead. In this case only minor modifications to the existing lead are 

introduced at each of the iterations of the drug design cycle. Many of 

these modifications may be either proposed from personal knowledge or 

derived by computer modeling. However, it is important to note that 

computational methods are still not reliable in predicting binding 

modes and affinities of ligands, mainly because of inaccuracies in force 

fields, limitations in dealing with ligand and target flexibility, and the 

lack of a reliable scoring functions, as well as the difficulties in treating 

solvent molecules. Therefore, even for seemingly minor modifications of 

the leads, it is still necessary to confirm the binding mode experimentally; 

there are countless examples in which the mode of binding 

significantly changes upon introduction of minor modifications to the 

original lead. 

VII. EXPERIENCE WITH STRUCTURE-BASED 

DRUG DESIGN 

Any summary of experience gained during the last 15 years in the area of 

structure-based drug design is in some way a work in progress, and clearly 

there is much that we still need to learn. 

A. Design Should Be Based on Liganded Structures 

Many proteins undergo considerable conformational change upon binding 

to their ligands. Initiating ligand design based on an unliganded structure 

may be misleading if that structure is of a protein that will change its 

conformation upon ligand binding. To be on the safe side, one should 

always start ligand design based on a liganded structure of the target 

protein. An example of a protein that undergoes large conformational 

change upon ligation is EPSP (5-enol-pyruvyl-3-phosphate) synthase. The 


unliganded structure [29] shows a large cavity at the active site (Fig. 7), 

much of which disappears upon ligation. Sometimes, different ligands lead 

to different conformational changes of the protein target, making the 

designing even more challenging. 

B. Design of Small Molecules to Interfere with 

Protein–Protein Interaction Requires the Structure 

of the Complex 

Most protein–protein interfaces are large hydrophobic surfaces. For 

example, the interface area between growth hormone and its receptor 

[30] is about 2100 A ˚ 2 . To rationally design a small molecule to interfere 

with such large surfaces is a considerable challenge that requires atomic 

details of the receptor surface, which may differ for unliganded and 

liganded forms. Generally, success in this arena is rare. Occasionally, 

protein–protein interactions consists of only a small number of contacts, 

such as the RGD interaction with its receptors [31]. In such a case, the 

Figure 7 Stereoview of the structure of EPSP synthase in its open conformation. 


design task becomes essentially a small-molecule–protein interaction 

problem and is much more likely to be successful. 

C. Allow for Flexibility in the Design of Enzyme 

Inhibitors to Assure Optimal Fit in an Often Rigid 

Active Site Cavity 

It is often very difficult to design a highly constrained ligand that complements 

and fits snugly in an enzyme active site. Although rigidity of the 

ligand is important to reduce entropy and to ensure greater affinity, it is 

often wise to initially introduce some flexibility to ensure proper fit in an 

often rigid active site. Much of this flexibility could be reduced as much as 

possible in later iterations of the drug design cycle. 

D. Synthetic Accessibility Is Essential 

It is important to design ligands that can be synthesized in a timely 

fashion from readily available or easily obtained starting material. Given 

that many potential drugs fail for reasons that have nothing to do with 

their binding affinity, it is important that one go through a design cycle as 

fast as possible to obtain feedback on the suitability of the designed 

ligands as drugs. 

E. Every Water Molecule Is Special 

Incorporation of the position of water molecules that are firmly bound to 

the protein can impart affinity and novelty to the designed ligand. A prime 

example is the design of a class of HIV protease cyclic urea inhibitors by 

DuPont scientists that incorporated a water molecule bound to both flaps 

of the enzyme into their ligand [32]. The crystal structure of the HIV 

protease–cyclic urea complex [32] shows the urea carbonyl oxygen substituting 

for the position of the water molecule. 

F. Fill Available Space and Maximize Interactions 

A major goal of ligand design should be to fill as much of the space in the 

binding site as possible without rendering the designed ligand too large. 

Ligands greater than 500 Da have lower probability of being orally 

bioavailable. It is also important to maximize both polar and nonpolar 

interactions with the protein. 


G. Beware of Crystal Contacts 

In the crystal, it is possible for a ligand to make important contacts with 

residues from a neighboring molecule producing an artificial mode of 

binding that is not possible in solutions. Thus it is important to analyze all 

crystal contacts in the vicinity of the ligand prior to proceeding with the 

design of new ligands. 

H. Use of Surrogate Enzymes Can Lead to Important 

Insights, But Optimization Requires the 

Target Protein 

When the target enzyme is difficult to obtain, related enzymes could be 

used to provide insights in the design of novel ligands. For example, papain 

was used to design a class of potent cathepsin K inhibitors [33] spanning 

both sides of the papain active site. However, fine-tuning these inhibitors to 

produce more potent ones required the use of the crystal structure of 

cathepsin K itself [34]. 

I. Iterative Design Is Essential 

It is a rarity that the first ligand to be designed is the final one. Thus, it is 

common to go through the structure-based design cycle (Fig. 2) several 

times with each class of inhibitors being designed. This iteration should 

continue until the ideal molecule that will be advanced to development has 

been identified. 

J. Solubility of Ligands Matters 

One of the bottlenecks associated with structure-based design is poor 

aqueous solubility of many ligands. If the ligands are insoluble in water, it 

is often difficult to form complexes under conditions of crystallization. 

Unlike the crystallization of small organic molecules, proteins must be 

crystallized from aqueous solutions or using solvents that are highly 

miscible with water. Therefore, it is sensible to introduce polar or charged 

groups to improve inhibitor solubility, making the target molecule more 

amenable to structural studies. 

K. No Substitute for Experience 

Structure-based drug design is no different from most other areas, in that 

experience counts greatly. 


L. Dedicated Molecular Biology and Protein 

Purification Groups Are Essential 

Protein crystallography often requires special constructs or mutants to 

facilitate crystallization; it also requires large quantities of highly purified 

protein. Thus to move forward in a timely fashion, it is important that an 

industrial structural biology group employ molecular biologists and 

individuals with expertise in protein purification. 

VIII. OUTLOOK 

Structure-based drug design is now an integral part of most if not all drug 

discovery programs. It is a given that structure-based design is part of each 

drug discovery effort whenever the target is a soluble protein. However, a 

large segment of targets—namely, membrane proteins, particularly Gprotein-coupled 

receptors—are excluded. It is hoped that this situation will 

be remedied in the near future. 

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2 Src Homology-2 Domains 

and Structure-Based, 

Small-Molecule Library Approaches 

to Drug Discovery 

Chester A. Metcalf III and Tomi Sawyer 

ARIAD Pharmaceuticals, Inc., Cambridge, Massachusetts, U.S.A. 

The elucidation of cell-receptor-associated signal transduction pathways 

by means of the tools of biochemistry and molecular genetics has 

resulted in the identification of a multitude of protein targets for 

therapeutic intervention (Table 1) [1]. The fact that many of these 

targets have x-ray crystallography and/or NMR spectroscopy to guide 

the syntheses of structurally biased single analogues and combinatorial 

libraries has ushered the pharmaceutical industry into a new era of drug 

discovery. Within cells there exists an enormously diverse data set of 

functional proteins and signaling pathways, involving both noncatalytic 

and catalytic processes, which are orchestrated through highly specific 

protein–protein interactions. In principle, such interactions can be 

disrupted or promoted, either directly or indirectly (via enzyme inhibition), 

through small-molecule intervention driven by structure-based 

methods. This chapter discusses the role of Src homology-2 (SH2) 

domains as mediators of protein–protein interactions in signal transduction, 

with a focus on the therapeutic implications of blocking the 

SH2 domain of the nonreceptor protein tyrosine kinase Src with 


Table 1 List of Possible Protein Targets for Therapeutic Intervention 

G-Protein-Coupled/Integrin 

Receptors 

Receptor/Nonreceptor 

Kinases/Phosphatases 

Transcription 

Factors/Proteases 

Angiotensin (AT1, AT2) Receptor tyrosine kinases NF-nB, STAT, NFAT, SMAD, CREB 

Bradykinin (B1, B2) Epidermal growth factor 

Cholecystokinin (CCKA) Fibroblast growth factor Proteases 

Gastrin (CCKB) Insulin Aspartic proteases 

Endothelin (ETA, ETB) Nerve growth factor Pepsin 

a–Melanotropin (MCR1) Platelet-derived growth factor Renin 

Adrenocorticotropin (MCR2) Cathepsins (D, E) 

Substance P (NK1) Nonreceptor tyrosine kinases HIV-1 protease 

Neurokinin-A (NK2) Src and Src family (Lck, Hck) 

Neurokinin-B (NK3) Abl, Syk, Zap-70 Serinyl proteases 

y-opioid (Enkephalin) Trypsin 

A-opioid (Endorphin) Receptor serine/threonine kinases Thrombin 

n-opioid (Dynorphin) Transforming growth factor Chymotrypsin-A 

Oxytocin Kallikrein 

Somatostatin (sst1–sst5) Nonreceptor serine/threonine kinases Elastase 

Vasopressin (V1A, V1B) cAMP-Dependent protein kinase Tissue plasminogen activator 

Neuropeptide-Y (Y1-Y5) Phosphoinositol-3-kinase (P13K) Factor Xa 

Calcitonin Cyclin-dependent kinases (CDKs) 


Adenosine (A1-A3) Mitogen-activated protein kinase Cysteinyl proteases 

Cathecholamine (a1, a2, h1-h3) Protein kinase C (PKC) Cathepsins (B, H, K, M, S, T) 

Histamine (H1, H2) Janus family kinases (JAKs) Proline endopeptidase 

Muscarinic acetylcholine InB family kinases (IKKs) Interleukin-converting enzyme 

Seratonin (5HT1-5HT7) Apopain (CPP-32) 

Melatonin (ML1A, ML1B) Receptor/nonreceptor phosphatases Picornavirus C3 protease 

Dopamine (D1, D2, D4, D5) Receptor tyrosine phosphatases Calpains 

g-Amino butyric acid (GABAB) CD45, LAR 

Leukotrienes (LTB4, LTC4, LTD4) Metallo proteases 

Nonreceptor tyrosine phosphatases Exopeptidase group 

Cell adhesion integrin receptors PTP1B, Syp Peptidyl dipeptidase-A (ACE) 

avh3 (Fibrinogen) Aminopeptidase-M 

aIIbh3 or gpIIaIIIb (Fibrinogen) Nonreceptor serine/threonine phosphatases Carboxypeptidase-A 

a5h1 (Fibrinectin) PP-1 

a4h1 (VCAM-1) Calcineurin Endopeptidase group 

VH1 Endopeptidases (24, 11, 24, 15) 

Stromelysin 

Gelatinases (A, B) 

Collagenase 

Source: Ref. 1. 


designed, nonpeptide small molecules. We highlight ARIAD’s approach 

to drug discovery by means of structure-based methods and parallel 

synthetic libraries to develop cell-active, in vivo effective inhibitors of Src 

SH2-dependent signal transduction pathways, leading to novel drugs for 

the treatment of osteoporosis. 

I. SIGNAL TRANSDUCTION AND PROTEIN–PROTEIN 

INTERACTIONS 

The network of protein–protein interactions that define signal transduction 

pathways in most cells originates at a cellular receptor and is 

triggered by the binding of specific external stimuli (e.g., growth factors, 

antigens, hormones). Such signal transduction pathways are then propagated 

within the cell to the nucleus resulting in specific gene activation 

and protein synthesis (Fig. 1) [2]. Listed in Table 2 are the modular 

domains [3] of various signal transduction proteins and the potential 

disease areas providing opportunity for therapeutic intervention through 

small-molecule inhibitory drugs [4]. For Src, these disease areas are 

osteoporosis and cancer. There are more than 50 known SH2-containing 

proteins, of which Src was the first to be identified [5]. The SH2 domain 

of Src consists of approximately 100 amino acids and binds cognate 

Figure 1 Representation of signal transduction pathways describing highly 

specific protein–protein interactions. 


Table 2 Signal Transduction Proteins as Potential Therapeutic Targets 

Protein (Domains) Disease area 

Src (SH3-SH2-kinase) Osteoporosis, cancer 

Hck (SH3-SH2-kinase) Immune disease, AIDS 

Syk (SH2-SH2-kinase) Allergy, asthma 

Zap70 (SH2-SH2-kinase) Autoimmune disease 

Syp (SH2-SH2-phosphatase) Anemia 

STATs (DNA binding-SH3-SH2) Inflammatory disease 

Grb2 (SH3-SH2-SH3) Cancer, chronic myelogenous leukemia 

p85/PI3K [SH3-SH2-SH2 (p85 subunit)] Cancer 

Bcr/Abl (SH3-SH2-kinase) Chronic myelogenous leukemia 


phosphotyrosine(pTyr)-containing proteins as well as synthetic peptides in 

a sequence-dependent manner [6]. In addition to the SH2 domain, Src 

possesses one SH3 domain (f 60 amino acids), which is characterized by 

its affinity for proline-rich sequences, and a bilobed tyrosine kinase 

catalytic domain (f 300 amino acids) containing N-terminal (NT) and 

C-terminal (CT) domains (Fig. 2) that specifically phosphorylates tyrosine 

residues of cognate substrate proteins. 

II. Src TYROSINE KINASE AND OSTEOPOROSIS 

Molecular insight into the protein conformation states of Src kinase has 

been revealed in a series of x-ray crystal structures of the Src SH3–SH2– 

kinase domain that depict Src in its inactive conformation [7]. This form 

maintains a ‘‘closed’’ structure, in which the tyrosine-phosphorylated 

(Tyr527) C-terminal tail is bound to the SH2 domain (Fig. 2). The x-ray 

data also reveal binding of the SH3 domain to the SH2–kinase linker 

[adopts a polyproline type II (PP II) helical conformation], providing 

additional intramolecular interactions to stabilize the inactive conformation. 

Collectively, these interactions cause structural changes within the 

catalytic domain of the protein to compromise access of substrates to the 

catalytic site and its associated activity. Significantly, these x-ray structures 

provided the first direct evidence that the SH2 domain plays a key role in 

the self-regulation of Src. 

The bone disease osteoporosis results when an imbalance occurs in 

the normal course of bone remodeling, a dynamic and highly regulated 


Figure 2 Depiction of the active (‘‘open’’) and inactive (‘‘closed’’) conformations 

of Src kinase based on the analysis of x-ray structures of c-Src tyrosine 

kinase crystallized in its inactive state [7]. The stabilization of the inactive 

conformation is influenced by multiple events including intramolecular binding 

of the tyrosine-phosphorylated C-terminus tail to the SH2 domain as well as 

interactions between the SH3 domain and the SH2–kinase linker. CT, C-terminal; 

NT, N-terminal. 

process that involves both bone degradation (resorption) and bone formation. 

Aberrantly high levels of bone resorption are associated with this 

disease, which effects a net decrease in bone density and volume, resulting 

in fragile, brittle bones that are subject to premature breaks and fractures 

[8]. The most compelling evidence that Src is intimately involved in bone 

remodeling comes from genetically engineered Src knockout mice. In these 

Src (–/–) mice, the only major phenotype observed is excessive bone 


formation; a condition termed osteopetrosis (the opposite of osteoporosis). 

This suggests that selective inhibition of Src, as a therapeutic treatment 

for osteoporosis, may shift the bone microenvironment from a state of 

perpetual bone degradation to one of normal bone turnover without 

deleteriously affecting other Src-associated cellular processes in the body. 

The rationale for Src’s involvement in bone processes becomes 

apparent when the Src knockout effects are examined at the cellular level 

of an osteoclast. Osteoclasts are multinucleated cells that are responsible 

for bone resorption. Two different osteoclasts, a wild-type (normal) and 

a Src (–/–) osteoclast, are shown schematically in Figure 3. The wild-type 

cell shows the characteristics of a bone-resorbing osteoclast, including a 

ruffled border and ‘‘pit’’ formed by the bone-degrading actions of an 

active osteoclast. These features are absent in the Src (–/–) knockout 

osteoclast, albeit they are still viable and adhere to bone. In 2000 

(Marzia et al. and Amling et al.) it was suggested that Src plays a 

negative regulatory role in osteoblasts (cells that are responsible for the 

Figure 3 The effect of an Src (–/–) knockout in mice as shown by differences in 

function and appearance of wild-type and Src-minus osteoclasts. The Src-minus 

osteoclasts lack the ruffled borders of a normal, resorbing osteoclast, but are 

viable and can adhere to bone. (From Ref. 8.) 


formation of bone) as shown by enhanced bone formation and osteoblast 

differentiation rates in Src (–/–) mice [8]. Together, these data 

provide complementary, mechanistic evidence to validate Src as a viable 

therapeutic target for the treatment of bone diseases such as osteoporosis. 

III. SH2 DOMAINS AND PHOSPHOPEPTIDE BINDING 

The question of whether ligand binding specificity exists among SH2 

domains was addressed, quite elegantly, by Cantley et al. [6,9], who used 

synthesized combinatorial libraries of pTyr-containing peptides. A majority 

of the SH2 binding affinity in pTyr-containing peptides can be 

attributed to a four-amino acid region represented by the sequence pTyr- 

Aaa-Bbb-Ccc. However, binding specificity exists in the three amino acids 

directly C-terminal to the pTyr (pY) group, referred to sequentially as 

pY+1 (Aaa), pY+2 (Bbb), and pY+3 (Ccc). The preferred pY+1, 

pY+2, and pY+3 amino acids for various SH2-containing proteins are 

listed in Table 3; the first amino acid listed for each position represents the 

most preferred. For Src SH2 this sequence is pTyr-Glu-Glu-Ile. Such 

sequence specificity among SH2-containing proteins provides a rationale 

for the differentiation of their associated signal transduction pathways 

in cells. 

The successful design of small molecules to interact with a protein 

binding surface is markedly enhanced by an understanding of the target’s 

three-dimensional structure, preferably in the context of a bound ligand. 

In this regard, early x-ray structures of pTyr-containing peptides bound 

to Src SH2 [10,11] paved the way for the discovery of peptide, peptidomimetic, 

and nonpeptide ligands and the determination of their complexed 

structures with Src SH2 [12–14] or the highly homologous Lck 

SH2 [15,16]. In a landmark paper, Waksman, Kuriyan, and their 

colleagues reported [11] the first x-ray structure of a high affinity phosphopeptide 

(Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-Ile-Tyr-Leu) bound Src 

SH2 (KD = 3–6 nM), which uncovered key protein interactions with the 

pTyr-Glu-Glu-Ile sequence. In particular, this x-ray structure shows the 

bound phosphopeptide oriented perpendicular to a central h sheet and 

interacting with two major binding regions of the Src SH2 domain, 

namely, one for the ligand pTyr (pY pocket) and another for the ligand 

Ile (pY+3 pocket), to provide what has been described as a ‘‘twopronged’’ 

binding mode. The pY pocket is characterized mainly by 


Table 3 SH2 Specificity for Phosphotyrosine-Containing Peptides 

-Asp-Gly-[pTyr-Aaa-Bbb-Ccc]-Ser-Pro- 

(pY)(pY+1)(pY+2)(pY+3) 

SH2 Domain Aaa Bbb Ccc 

Group 1A a 

Src Glu, Asp, Thr Glu, Asn, Tyr Ile, Met, Leu 

Lck Glu, Thr, Gln Glu, Asp Ile, Val, Met 

Group 1B a 

Abl Glu, Thr, Met Asn, Glu, Asp Pro, Val, Leu 

Syk (N-SH2) Gln, Thr, Glu Glu, Gln, Thr Thr 

Syk (C-SH2) Thr Thr Ile, Leu, Met 

Grb2 Gln, Tyr, Val Asn Tyr, Gln, Phe 

Group 3 b 

PLCg (N-SH2) Leu, Ile, Val Glu, Asp Leu, Ile, Val 

PLCg (C-SH2) Val, Ile, Leu Ile, Leu Pro, Val, Ile 

p85 (N-SH2) Met, Ile, Val — Met 

SHC Ile, Glu, Tyr — Ile, Leu, Met 

a Group 1 contains Tyr or Phe at hD5. 

b Group 3 contains Ile, Cys, or Leu at hD5. 

Sources: Refs. 6, 9. 

electrostatic interactions, while the pY+3 pocket involves interactions 

that are mostly hydrophobic. 

Figure 4 represents the specific Src SH2 binding interactions with 

pTyr-Glu-Glu-Ile sequences, as interpreted from x-ray structures [10,11]. 

The major intermolecular interactions in the pY pocket involve the 

phosphate oxygens of the ligand pTyr side chain with the conserved basic 

residues Arg158 and Arg178 of Src SH2. It is noted that Arg178 mutation 

results in essentially a total loss of binding affinity [17]. Additional 

intermolecular hydrogen-bonding interactions are also observed with 

Ser180, Thr182, and the backbone NH of Glu181, whereas a hydrophobic 

contact occurs between the alkyl side chain of Lys206 and the phenyl ring 

of the ligand pTyr residue. The two adjacent glutamic acid residues 

(pY+1 and pY+2) form relatively weak interactions (electrostatic and 

hydrophobic) with the protein, albeit their extend side chain conformations 

(oriented away from each other) serve to align and rigidify the 

peptide backbone. This is an important feature from a drug discovery 

perspective and can be used in the design of rigid, nonpeptide templates 


Figure 4 Representation of the binding interactions involving the phosphopeptide 

motif pTyr-Glu-Glu-Ile with Src SH2 as interpreted from complexed x-ray 

structures [10,11]. The binding regions of the protein, including the major pY and 

pY+3 pockets, are represented by their key binding residues. Also included are 

the observed structural waters and their interactions with the pY+1 Glu and pY+3 

Ile phosphopeptide residues. 

to advance Src SH2 inhibitors (see later: Sec. IV, Lead Discovery and 

Combinatorial Chemistry). 

The only direct ligand–protein hydrogen bond contact involves the 

backbone NH of the pY+1 Glu with the carbonyl oxygen of the His 204 

residue. In addition to the hydrophobic interactions involving the Ile 

phosphopeptide residue and the pY+3 pocket, there exist potential 

hydrogen-bonding possibilities from Tyr205, Ile217, and a buried 

Tyr233 residue. Finally, two structural water molecules provide hydrogen-bonding 

networks between the pY+1 Glu (CO) and pY+3 Ile (NH) 

phosphopeptide residues, and the Lys206 (NH) and Ile217 (CO) Src SH2 

protein residues, respectively. Such structural waters act as drug design 

elements to increase binding affinity (through favorable entropic contributions) 

and can be exploited by small molecules that bind to or displace 

them (see later: Sec. VI, Structure-Based, Small-Molecule Libraries to 

Explore Src SH2 Binding). 

The importance of the pTyr group for SH2 binding is counterbalanced 

by the biological instability of the phosphate group to cellular 


Figure 5 List of pTyr mimics containing nonhydrolyzable and reduced charge 

functionality, which were explored in the context of a pTyr-Glu-Glu-Ile peptide. 

(From Ref. 12.) 

phosphatases as well as low cellular permeability posed by the highly 

charged phosphate group [18]. These issues have prompted the pursuit of 

pTyr mimics to discover cellulary active inhibitors. In a comparative 

binding study involving pTyr mimics, in the context of a pTyr-Glu-Glu- 

Ile sequence, researchers explored the ability of a variety of functional 

groups to act as pTyr replacements (Fig. 5) [12]. The highest affinity, 

nonhydrolyzable pTyr replacement was found to be the F2Pmp (difluorophosphonomethyl 

phenylalanine) group [19]. Although some of the 

aforementioned pTyr replacements represent nonhydrolyzable moieties, 

the design of a stable pTyr mimic providing both high affinity and adequate 

cell permeability has remained challenging [20]. 

IV. LEAD DISCOVERY AND COMBINATORIAL 

CHEMISTRY 

The integration of structural biology, drug design (molecular modeling 

and ‘‘druglike’’ assessment), and synthetic chemistry to discover novel 

small-molecule leads follows the general iterative process outlined in 

Figure 6. Available structural knowledge is used to design pharmaceutically 

driven compounds that will bind a desired protein target; these 


Figure 6 The iterative drug discovery process integrating structural biology, 

drug design, synthetic chemistry, biological testing, and additional input from 

other related research areas. 

compounds are then synthesized and tested in the appropriate assays. The 

biological data are analyzed in the context of available (x-ray or NMR) 

structural information to impact the design of the next series of analogues. 

This process is repeated until a lead compound or series of compounds 

possessing the desired biological activities are obtained. 

The database of available structural information during ARIAD’s 

initial investigation into compounds targeting Src SH2 was limited; cases 

involving ligand complexes utilized only peptide molecules [21]. Motivated 

by an interest to develop orally active Src inhibitors (i.e., nonpeptides) we 

adopted an exploratory approach to small-molecule lead discovery, using a 

combinatorial chemistry strategy. Combinatorial libraries were biased 

with a common phenyl phosphate group and systematically engineered 

with diversity elements (selection guided by modeling) to probe the protein 

surface for existing and new binding interactions (Fig. 7). Solid phase array 

synthesis encompassing a novel phosphate ester linker strategy [22] was 


Figure 7 A novel, phosphate ester linking strategy [22] was used in the synthesis 

of phenyl phosphate–containing compound libraries, accomplished in 96deepwell 

reaction blocks [23]. Rigid, nonpeptide templates (A-group) and 

pY+3 substituents (B-group) satisfied the diversity sites of the molecules. 

used to construct the compound libraries in a 96-deepwell plate format 

[23]. The diversity elements of the molecules included nonpeptide templates 

(A-group) and pY+3 substituents (B-group). The A-group diversity 

elements were typically rigid and provided access to both the pY and 

pY+3 pockets (in a manner similar to the aforementioned Glu-Glu 

sequence) as well as directionality for each attached substituent. Binding 

interactions targeting the pY+3 pocket were explored through hydrophobic 

B-group diversity elements. Finally, diversity building blocks 

were chosen to target final products in the molecular weight range of 

500 to 600. Compounds were screened in a high throughput, fluorescent 

polarization (FP) binding assay [24], using estimated concentrations 

(relative to a 50 mM DMSO product stock solution assuming 100% 

synthetic conversion). To verify final product formation, we performed 

qualitative analysis for all compounds using electrospray (+/–) mass 

spectroscopy. An HPLC peak area purity assessment was also conducted 

for selected compounds. 

V. SOLID-PHASE PARALLEL SYNTHESIS AND 

NONPEPTIDE PHENYL PHOSPHATE LIBRARIES 

The combinatorial construction of compound libraries in 96-deepwell 

plates is efficiently accomplished by adhering to the two-dimensional grid 


pattern of the plate. For example, if different diversity elements are 

added across the rows of the plate (one diversity element, repeated 8 

times, per row), with the 12 columns housing the second set of diversity 

elements, 96 discrete compounds (e.g., 8 A-group 12 B-group = 96 

compounds; Fig. 7) will result. This format permits the rapid synthesis of 

relatively large structurally biased libraries by systematically combining 

sets of diversity groups. 

To streamline plate synthesis, we developed with Cyberlab, Inc. [25] a 

custom high throughput organic synthesizer designed to process the 96deepwell 

reaction blocks (Fig. 8). This instrument was constructed to 

tolerate a wide range of chemistries; therefore, all liquid contacts (syringes, 

needles, tubes, and valves) are made of glass, stainless steel, or Teflon. 

Coaxial tip needles with N2 inlets (connected to a bubbler) allow inert 

dispensing and withdrawal of liquid reagents from the closed vessels 

without excessive negative or positive pressure buildup. The instrument 

head, which can access all positions on the deck, is fitted with single-needle 

and four-needle probes. The tandem use of both needle probes facilitates 

the transfer (via 5 mL syringe pumps, not shown) of all reaction intermediates 

from the reagent vials (left side) to the resin-containing 96deepwell 

reaction blocks (right side). 

The reaction block, which provides a fully enclosed reaction 

environment (Teflon, polypropylene, and silicone rubber seals) is a 

slightly modified version of a design first disclosed by Sphynx Pharmaceuticals 

[23]. Figure 9 shows the reaction block and the reagent 

vials (100, 30, and 10 mL sizes) in their fully assembled and disassembled 

states. Holes at the bottom of the wells of the 96-deepwell 

polypropylene plate (sealed in fully assembled reaction block) allow the 

reaction solutions to be removed from the wells (via a separate vacuum 

plenum) and the functionalized resin (retained by Teflon frits) to be 

washed with solvents. 

The use of the phenyl phosphate group as both a solid support 

attachment site and a crucial binding element represents what has been 

referred to as a ‘‘pharmacophore-linking’’ strategy [26]. We explored a 

variety of phenyl phosphate tether functionalities to provide resins varying 

in substitution pattern and in chemical flexibility (Scheme 1 and Table 4) 

[22]. All phenyl phosphate resins were synthesized in batch quantities of 

20 g or more. Resin synthesis began with the addition of either p-methoxybenzyl 

alcohol or benzyl alcohol to commercially available bis(diisopropylamino)chlorophosphine, 

followed by addition of the diversity phenol 

[(R 1)-OH, DIAT (diisopropylamino tetrazole)]. Displacement of the 


Figure 8 High throughput organic synthesizer developed in collaboration with 

Cyberlab, Inc. [25] and designed to process the 96-deepwell reaction blocks. The 

instrument is capable of tolerating a wide range of chemistry (liquid contacts are 

glass, stainless steel, or Teflon) and accomplishes the transfer of reagents with 

coaxial tip (N2 inlets) single-needle and four-needle probes. 

remaining diisopropylamino group from 1 with Wang resin and oxidation 

with 4-methylmorpholine N-oxide (NMO) provided the protected phenyl 

phosphate resins 2 and 3 in excellent yields, as shown in Table 4. Two types 

of functionality, namely, protected carboxy and amino groups, differentiated 

the starting phenols. Reaction schemes demonstrating compound 

synthesis using both phenol types are shown in Scheme 2 [22]. Mild Fmoc 

deprotection (1% DBU/DMA) of resin 2a and amide formation using 

standard coupling conditions [TBTU, DIEA, p-(CO2H)PhCH2NH- 

Fmoc)] resulted in attachment of the first diversity element to provide 

resin product 4. A second deprotection followed by a double, one-pot 


Figure 9 The 96-deepwell reaction block and the reagent vials (100, 30, and 10 

mL sizes) used in the organic synthesizer in their fully assembled and 

disassembled states. 

Scheme 1 


Table 4 Yields and Loading of Phenylphosphate Resins 

Resin R 1 Loading (mmol/g) Yield (%) 

2a p-(FmocNHCH 2CH 2)Ph 0.659 93 

2b p-(FmocNHCH2)Ph 0.637 89 

2c m-(FmocNHCH2)Ph 0.627 88 

2d p-(Allyl-O 2CCH 2CH 2)Ph 0.730 92 

2e p-(Allyl-O2CCH2)Ph 0.672 84 

3f m-(Allyl-O2CCH2)Ph 0.807 98 

3g p-[ p-(FmocNHCH 2)PhO]Ph 0.552 81 

3h p-(Allyl-O 2CCH=CH)Ph 0.535 66 

3i m-(Allyl-O2CCH=CH)Ph 0.796 98 


Scheme 2 Abbreviations: DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMA, 

N,N-dimethylacetamide; TBTU, O-benzotriazole-1-yl-N,N,NV,NV-tetramethyluronium 

tetrafluoroborate; DIEA, diisopropylethylamine; RA, reductive amination; 

TFA, trifluoroacetic acid; DCM, dichloromethane. 


eductive amination [Na(OAc)3BH, 2-ethylbutyraldehyde] provided the 

fully coupled compound with a branch point at the second diversity 

site. Mild conditions (30% TFA/CH2Cl2) to cleave the final compound 

from the solid support as well as to remove the p-methoxybenzyl protecting 

group resulted in the isolation of compound 5 in 86% HPLC purity, 

following in vacuo concentration. For the synthesis of compound 7, the 

allyl ester of resin 2d was deprotected under palladium-mediated conditions, 

followed by amide coupling [TBTU, DIEA, m-(NH 2CH 2) PhCO 2allyl] 

to generate the functionalized phenyl phosphate resin 6. A second 

deprotection and coupling [TBTU, DIEA, NH(Me)CH2Ph] provided the 

bisamide resin-bound compound, which was cleaved and isolated as 

described earlier to yield compound 7 in 66% HPLC purity. 

The compound types synthesized by using the foregoing combinatorial 

approach are represented in Figure 10. Variations in functional 

group connectivity (e.g., amides, olefins, sulfonamides) reflect the wide 

range of chemistry that was pursued in the generation of these libraries. 

Some bifunctional A-group and monofunctional B-group diversity elements 

used in the coupling reactions are shown in Figure 11. Alkyl 

and aryl phosphate ester groups (R; see Fig. 10) were also explored to 

Figure 10 Representations of some of the compound types synthesized in the 

nonpeptide phenyl phosphate libraries. 


investigate the binding consequences of reduced charge at the phosphate 

group. Initial Src SH2 screening produced hits, which were resynthesized 

and then retested in the binding assay. Some of the higher affinity 

compounds are shown in Figure 12. Although more than 10,000 compounds 

were produced by this methodology, only marginal binding 

affinities and no high-resolution x-ray or NMR structures were achieved; 

the poor aqueous solubility and undesirable physical properties of the 

molecules are likely to have hampered these efforts. At this point a decision 

was made to pursue a much more structure-based approach. Compounds 

Figure 11 List of some of the molecular diversity building blocks used in the 

construction of the nonpeptide phenyl phosphate libraries. 


Figure 12 Resynthesized library hits identified from the high throughput 

fluorescence-polarization assay along with their Src SH2 binding data. 


lending themselves to possible x-ray and/or NMR co structure determination 

were emphasized. 

VI. STRUCTURE-BASED, SMALL-MOLECULE 

LIBRARIES TO EXPLORE Src SH2 BINDING 

Refocusing our drug-discovery strategy prompted us to revisit the initial 

lead compound, pTyr-Glu-Glu-Ile. It was clear that to generate high 

affinity, small-molecule compounds for Src, we would likely need to 

maintain the key binding interactions of the pTyr-Glu-Glu-Ile motif, as 

well as to explore molecules capable of mimicking or interacting with the 

structural waters found in the Src SH2-phosphopeptide complexed x-ray 

structure. A template would be required that allowed access to both 

pockets (pY and pY+3), mimicking the ‘‘two-pronged’’ binding mode 

of pTyr-Glu-Glu-Ile. Noteworthy in this regard, a novel, de novo designed 

nonpeptide 8 was disclosed [14] with comparable binding to Ac-pTyr- 

Glu-Glu-Ile-NH2 (phosphopeptide 9) (Fig. 13). Significant interactions 

involving the benzamide functionality were revealed in an x-ray structure 

of 8 bound to Src SH2 [14]. In addition to interacting with several key sites 

of Src SH2 (e.g., the pY/pY+3 pockets and the CO of His204), this 

compound displaces both structural water molecules and makes a direct 

hydrogen bond contact with the backbone NH of Lys206 through its 

benzamide CO moiety. The effect of this carboxamide group on Src SH2 

binding is demonstrated by the related compounds 10 and 11 [14], in which 

the desamide compound 11 binds with over 15-fold lower affinity than 10 

(Fig. 13). ARIAD’s strategy was to utilize this high affinity benzamide 

template to gain a better understanding of nonpeptide interactions with Src 

SH2, and then to advance a database of structure–activity relationships 

(SARs) to ultimately develop novel, proprietary Src SH2 inhibitors. 

Subsequent to the disclosure of compound 8, a second-generation, higher 

affinity compound, containing a methylated benzamide template in the 

context of a pTyr group, was reported [27]. A literature procedure [27] was 

used to synthesize this compound (12, AP21733) [16], and a 2.5 A ˚ x-ray 

crystal structure of Lck SH2 (S164C), a protein homologue of Src SH2, 

complexed with AP21733, was obtained (M. H. Hatada, unpublished 

results). The proposed S-configuration of the benzylic methyl stereocenter 

of AP21733 was confirmed through independent asymmetric synthesis 

[28]. The Lck SH2–nonpeptide structure reveals adherence to the historical 

pTyr-Glu-Glu-Ile interactions in the pY pocket and shows carboxamide 


Figure 13 Series of de novo designed nonpeptides containing a benzamide 

template (exemplified by compound 12, AP21733) designed to interact favorably 

with Src SH2 and specifically to displace structural waters found in complexed 

Src SH2 structures [14,27]. The Src SH2 binding IC 50 is shown for each 

compound, as well as a comparative IC50 for Ac-pTyr-Glu-Glu-Ile-NH2 

(compound 9). 

contacts with Lys182 (206 in Src) and Ile193 (217 in Src). The phenyl ring 

of the benzamide template also forms favorable stacking interactions with 

Tyr181 (205 in Src). Although the cyclohexylmethyl group interacts with 

the pY+3 pocket, the contacts are primarily surface type and do not 

extend as deeply into the pocket as the Ile of pTyr-Glu-Glu-Ile. Consequently, 

SAR exploration of the pY+3 pocket, which had not been 

rigorously studied with nonpeptide (peptidomimetic) small molecules 

[13,14], became the first objective to be investigated. 

Parallel synthesis provides the means of rapidly preparing discrete 

analogues for both lead generation and lead optimization strategies, 

which makes it an attractive option for developing compound databases 

for therapeutic targets. Furthermore, the incorporation of structure-based 

methods into the design and evaluation of parallel synthetic libraries has 

proven to be a successful strategy for integrating the two drug discovery 

technologies [29]. For the synthesis of the benzamide-containing compounds, 

we devised a hitherto unreported solid phase, parallel synthetic 


oute focusing on pY+3 derivatives based on compound 13 and AP21733 

[30]. Our synthetic philosophy adopts an integrated solid and solution 

phase strategy that differs from the traditional unidirectional approach by 

recognizing the strengths and limitations of each synthetic method and 

then devising a route accordingly (Fig. 14) [31]. In addition, this strategy 

provides chemical flexibility to incorporate, within the compound’s 

molecular design, the necessary functional group complexity dictated by 

our structure-based methods. The importance of the carboxamide group 

guided our decision to exploit this functionality both as a solid support 

attachment site and as a conserved binding element. A Rink amide linkage 

was chosen to provide facile coupling of the template, via its benzoic acid, 

and eventual generation of the critical benzamide binding moiety upon 

cleavage from the solid support. The protected salicylic acid template 14 

(synthesized using a modification of the solution phase literature procedure) 

[27] was coupled to Rink amide AM resin by means of standard 

protocols (EDC/HOBt) to provide the benzamide-linked resin 15 

Figure 14 Parallel synthetic approaches demonstrating a traditional (unidirectional) 

strategy and a multifaceted, integrated strategy; the latter utilizes both 

solid and solution phase reactions. 


(Scheme 3) [30]. The pY+3 diversity alcohols (R1)-OH (Fig. 15) were 

attached to the template through a Mitsunobu coupling to provide ether 

derivatives of 16. Palladium-mediated Alloc deprotection followed by 

amide formation using the phosphate-ester-containing diversity acids 

(R2)-CO2H provided the fully coupled resin-bound products of 17. 

Cleavage from the resin with 95% TFA/H2O, which also afforded benzyl 

phosphate deprotection, followed by reversed-phase (RP) semipreparative 

Scheme 3 Abbreviations: EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide)hydrochloride; 

HOBt, 1-hydroxybenzotriazole; DEAD, diethyl 

azodicarboxylate. 


Figure 15 The diversity alcohols (R1)-OH and carboxylic acids (R2)-CO2H used 

to synthesize compounds represented by 18 and 19. (From Ref. 30.) 

Table 5 Src SH2 Binding (FP) for Analogs of Compound 18 



HPLC purification generated the final compounds represented by 18 

(mixture of diastereomers) and 19. 

The selection of the pY+3 diversity R1 groups was guided by a FLO 

docking program [32], utilizing 800 commercially available alcohols 

(prefiltered by MW, H-bond donors, and reactive groups outside the 

OH). The R1 groups were computationally incorporated [33] into the 

benzamide template, docked into our Src SH2 binding site model [34], and 

then rank-ordered according to favorable fit. The final list of alcohols was 

Figure 16 The predicted binding mode of compound 23 in the pY+3 pocket of 

the Src SH2 model. The branch point in the pY+3 bisallyl group allows favorable 

binding interactions to occur. (From Ref. 30.) 


selected according to predicted binding as well as the ability of the R1 

group to impart beneficial properties to the molecule as related to low 

molecular weight, increase solubility, and other factors. 

Table 5 contains the Src SH2 binding results for a selected set of 

pY+3-modified nonpeptide analog. Relative to compound 20, which was 

synthesized by means of our solid phase method to act as an internal 

standard, increases in binding affinity appeared to track the degree of 

hydrophobicity at the R 1 group as demonstrated by compounds 21 

(methyl) and 22 (isopropyl). From a drug design perspective, the result 

of 22 is significant because a four-carbon reduction took place, relative to 

the cyclohexylmethyl group (MW decrease by 54), without greatly compromising 

the binding affinity (four-fold). 

An extension of the a-branch point of the isopropyl group to a 

bisallyl resulted in the highest affinity analog, compound 23. Inspection of 

the docked structure of 23 in our Src SH2 model reveals how the branch 

point allows one allyl side chain to hug the surface of the protein, while the 

other is able to extend deeply into the pY+3 pocket (Fig. 16). A significant 

decrease in binding affinity occurs with the incorporation of a morpholine 

group, as exemplified by compound 24. Presumably, this result reflects an 

incompatibility of the positively charged morpholine group (at pH 7.2 of 

the binding assay) in the hydrophobic pY+3 binding pocket of the Src 

SH2 domain; structurally, the pY+3 pocket according to our Src SH2 

model accommodates this compound. 

VII. DISCOVERY OF AN IN VIVO EFFECTIVE Src 

SH2 INHIBITOR 

The next logical step in the progression to a cellularly active Src SH2 

inhibitor was to incorporate a high affinity, biologically stable pTyr 

mimic into the benzamide template. Drug design efforts at ARIAD led to 

a novel Src SH2 inhibitors containing 4-diphosphonomethylphenylalanine 

(Dmp), namely, compound 25 (AP21773; Fig. 17) [16]. The design 

concept for the Dmp group evolved from a 1.5 A ˚ x-ray structure of Src 

SH2, crystallized from citrate buffer, that fortuitously contained a citrate 

molecule bound in the pTyr pocket. The x-ray structure reveals a number 

of additional hydrogen bonds that citrate makes compared with a pTyr 

group; this inspired the design of the Dmp moiety as a novel mimic of the 

citrate interactions. Armed with these designed hydrogen bond contact 


Figure 17 Src SH2 binding IC50 (Fp) for compound 25 (AP21773), which 

contains a bone-targeted, 4-diphosphonomethylphenylalanine (Dmp) pTyr 

mimic. (From Ref. 16.) 

groups, we expected the Dmp to bind with greater affinity than pTyr, and 

the Src SH2 binding results for AP21773 (Dmp) and AP21733 (pTyr) 

confirm this prediction (Figs. 13 and 17). X-ray and NMR structural 

studies involving AP21773 [16] verify these additional Dmp-related 

contacts in the pTyr pcket, as well as other key Src SH2 interactions 

observed earlier with this benzamide class as already discussed. The Dmp 

moiety not only increases Src SH2 binding affinity, but also provides a 

mechanism for tissue selectivity by targeting bone [16,35]. This targeting 

feature provides a higher local concentration of compound on bone than 

Figure 18 Solid phase synthetic scheme and molecular diversity groups for 

compound 27. (From Ref. 36.) 


Table 6 Src SH2 Binding (FP), Rabbit Pit, and Rat TPTX Data for Analogs of 

Compounds 27 and 35 (AP22209) 


in solution, which in addition increases the amount of Src inhibitor 

delivered to the resorbing osteoclasts associated with bone. Compounds 

containing the Dmp group, including AP21773, also bind to hydroxyapatite 

(data not shown), a major component of bone [16]. 

Building on the SAR information obtained from the pY+3 study, 

we focused on improving binding affinity and cellular potency by means of 

structure-based, parallel synthesis. A resin-bound, enantiomerically 

enriched benzamide template 26 (Fig. 18) [28] was synthetically elaborated 

in a manner similar to that described in Scheme 3 to provide the desired 

Dmp-containing products. A total of 22 structurally biased analogues of 

27 were generated (not all combinations synthesized) having specific R1 

and R2 groups as shown in Figure 18 [36]. Table 6 shows the SAR results 

for a selected series of the benzamide analogues. Similar to the earlier 

study, increasing hydrophobicity at the pY+3 position leads to increased 

binding affinity, as demonstrated by compounds 28 to 30. Interestingly, 

the overall effect on Src SH2 binding of the 3-pentyl group of compound 

30 appears to be similar to that of the cyclohexylmethyl group of 

AP21773, although the latter group contains two more carbon atoms. 

All the derivatives show an approximately 5- to 10-fold reduction in Src 

SH2 binding affinity with no substitution (R2 = H, compounds 31–34) at 

Figure 19 Solid-phase synthetic scheme and molecular diversity groups for 

compound 36. 


the R2 position. Finally, in an effort to select compounds for testing in an 

in vivo thyroparathyroidectomized (TPTX) animal model [37,38], Src SH2 

inhibitors were evaluated in a cell-based resorption assay mediated by 

rabbit osteoclasts. A potent compound, 35 (AP22209; Table 6), was discovered 

and showed significant bone resorption inhibition in test animals 

(55% inhibition at 25 mg/kg b.i.d.), thus providing in vivo validation for 

an Src SH2 inhibitor (C. A. Metcalf III, unpublished results). 

A recent series of proprietary, nonpeptide Src SH2 inhibitors synthesized 

by our solid phase, parallel synthetic method is outlined in Figure 19. 

This inhibitor series was based on a set of compounds disclosed earlier 

[35,39,40] and containing a novel, high-affinity bicyclic benzamide template 

designed to interact favorably with the hydrophobic Tyr205 Src SH2 

protein residue. A bone-targeting, 3,4-diphosphonophenylalanine (Dpp) 

mimic of pTyr was also incorporated [35,40]. The Dpp moiety can be 

correlated to both pTyr and citrate (Fig. 20). The biological data for the 

library analogs of 36 will be described elsewhere. 

Figure 20 Representation of the design rationale for two novel, bone-targeting 

pTyr mimics, Dmp and Dpp, relative to an x-ray structure [16] of citrate 

complexed with Src SH2. 


VIII. CONCLUSION 

The increasing number of therapeutic targets available to drug discovery 

programs has challenged chemists to devise new and efficient strategies for 

the advancement of lead compounds to clinical candidate status. One 

evolving approach, as described in this chapter, is the integration of 

synergistic technologies (e.g., structure-based drug design and combinatorial 

chemistry) into a focused program that emphasizes the strengths of 

each individual method. We have used this philosophy to direct our Src 

SH2 program toward achieving novel proprietary Src SH2 inhibitors such 

as AP22209, which exhibit promising antiresorptive activity both in an in 

vivo animal model and in cell-based osteoclast assays. The use of structure-based, 

small-molecule libraries allowed us to rationally design compounds 

relative to predicted binding interactions, while taking advantage 

of parallel synthesis to rapidly advance lead optimization. By adopting a 

synthetic strategy that utilizes both solid and solution phase chemistries, 

we were able to achieve the necessary chemical purity and diversity for 

SAR interpretation at all stages of the drug discovery process. This 

integrated drug design and combinatorial chemistry strategy is currently 

being adapted to other drug discovery programs at ARIAD. 

ACKNOWLEDGMENTS 

The authors thank all our colleagues at ARIAD Pharmaceuticals, 

including Chi Vu, Virginia Jacobsen, Michael Yang, William Shakespeare, 

Regine Bohacek, Joseph Eyermann, Berkley Lynch, Shelia 

Violette, and Manfred Weigele, and especially Mayumi Uesugi, Vaibhav 

Varkhedkar, and Chad Haraldson, whose contributions were significant 

to the success of this work. We also thank Chris Stearns for her help with 

the figures, David Dalgarno for his editorial suggestions, and Jay 

LaMarche for allowing us to buy all our expensive toys. 

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1994; 14:2777–2785. 

10. Waksman G, Kominos D, Robertson SR, Pant N, Baltimore D, Birge RB, 

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Nature 1992; 358:646–653. 

11. Waksman G, Shoelson SE, Pant N, Cowburn D, Kuriyan J. Binding of a 

high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal 

structures of the complexed and peptide-free forms. Cell 1993; 72:779–790. 

12. Gilmer TM, Rodriguez M, Jordan S, Crosby R, Alligood KJ, Green M, 

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B, Akamatsu M, Shoelson SE, Wolf G, Giorgetti-Peraldi S, Yan X, Roller 

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Chem 1995; 38:4270–4275. 

13. Plummer MS, Holland DR, Shahripour A, Lunney EA, Fergus JH, Marks 

JS, McConnell P, Mueller WT, Sawyer TK. Design, synthesis, and cocrystal 

structure of a nonpeptide Src SH2 domain ligand. J Med Chem 1997; 

40:3719–3725. 

14. Lunney EA, Para KS, Rubin JR, Humblet C, Fergus JH, Marks JS, Sawyer 

TK. Structure-based design of a novel series of nonpeptide ligands that bind 

to the pp60 c-src SH2 domain. J Am Chem Soc 1997; 119:12471–12476. 

15. Eck MJ, Shoelson SE, Harrison SC. Recognition of a high-affinity 

phosphotyrosyl peptide by the Src homology-2 domain of p56 lck . Nature 

1993; 362:87–91. 

16. Bohacek RS, Dalgarmo DC, Hatada M, Jacobsen VA, Lynch BA, Macek KJ, 

Merry T, Metcalf CA III, Narula SS, Sawyer TK, Shakespeare WC, Violette 

SM, Weigele M. X-ray structure of citrate bound to Src SH2 leads to a high 

affinity, bone-targeted Src SH2 inhibitor. J Med Chem 2001; 44:660–663. 

17. Bibbins KB, Boeuf H, Varmus HE. Binding of the Src SH2 domain to 

phosphopeptides is determined by residues in both the SH2 domain and the 

phosphopeptides. Mol Cell Biol 1993; 13:7278–7287. 

18. For a discussion on the use of prodrugs to address bioreversibility and cell 

permeability for inhibitors of Src SH2, see: Rickles RJ, Henry PA, Guan W, 

Azimiora M, Shakespeare W, Violette S, Zoller MJ. A novel mechanismbased 

mammalian cell assay for the identification of SH2-domain-specific 

protein–protein inhibitors. Chem Biol 1998; 5:529–538, and Stankovic CJ, 

Surendran N, Lunney EA, Plummer MS, Para KS, Shahripour A, Fergus 

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in the selectivity and cellular uptake of SH2 domain ligands. 

Bioorg Med Chem Lett 1997; 7:1909–1914. 

19. Burke TR Jr, Smyth M, Nomizu M, Otaka A, Roller PP. Preparation of 

fluoro- and hydroxy-4-(phosphonomethyl)-D,L-phenylalanine suitably protected 

for a solid-phase synthesis of peptides containing hydrolytically stable 

analogues of O-phosphotyrosine. J Org Chem 1993; 58:1336–1340. 

20. For recent papers describing the use of pTyr mimics in the context of Src 

and Lck SH2 inhibitors, see: (a) Kawahata N, Yang MG, Luke GP, 

Shakespeare WC, Sundaramoorthi R, Wang Y, Johnson D, Merry T, 

Violette S, Guan W, Bartlett C, Smith J, Hatada M, Lu X, Dalgarno DC, 


Eyermann CJ, Bohacek R, Sawyer TK. A novel phosphotyrosine mimetic 

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design of nonpeptide inhibitors of pp60 src SH2 domain. Bioorg Med Chem 

Lett 2001; 11:2319–2323. (b) Lesuisse D, Deprez P, Bernard D, Broto P, 

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and rational drug design. Application to the discovery of nanomolar Src 

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DR, Ferland J-M, Gauthier J, Ghiro E, Gillard J, Gorys V, Poirier M, 

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nonhydrolyzable phosphate replacements. J Med Chem 1999; 

42:1757–1766. 

21. For examples of x-ray structures of nonpeptide compounds complexed with 

Src SH2, see Refs. 13 and 14, and for Lck SH2, see Ref. 16. 

22. Metcalf CA III, Vu CB, Sundaramoorthi R, Jacobsen VA, Laborde EA, 

Green J, Green Y, Macek KJ, Merry TJ, Pradeepan SG, Uesugi M, 

Varkhedkar VM, Holt DA. Novel phosphate ester-linked resins: the solidphase 

generation of phenyl phosphate-containing compounds for SH2 

inhibition. Tetrahedron Lett 1998; 39:3435–3438. 

23. Meyers HV, Dilley GJ, Durgin TL, Powers TS, Winssinger NA, Zhu H, 

Pavia MR. Multiple simultaneous synthesis of phenolic libraries. Mol 

Diversity 1995; 1:13–20. 

24. Lynch BA, Loiacono KA, Tiong CL, Adams SE, MacNeil IA. A 

fluorescence polarization based Src-SH2 binding assay. Anal Biochem 

1997; 247:77–82. 

25. Cyberlab, Inc., Brookfield, CT. 

26. Backes I, Ellman JA. Solid support linker strategies. Curr Opin Chem Biol 

1997; 1:86. 

27. Lunney EA, Para KS, Plummer MS, Prasad JVNV, Saltiel AR, Sawyer TK, 

Shahripour A, Singh I, Stankovic CJ. Compounds, composition and 

methods for inhibiting the binding of proteins containing an SH2 domain 

to cognate phosphorylated proteins. PCT Int Appl WO97/12903, 1997. 

28. Luke GP, Holt DA. Synthesis of (S)-5-(1-aminoethyl)-2-(cyclohexylmethoxy) 

benzamide. Tetrahedron: Asymmetry 1999; 10:4393–4403. 

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TC, Sarohia R, Leost M, Meijer L, Schultz PG. Synthesis and application of 

functionally diverse 2,6,9-trisubstituted purine libraries as CDK inhibitors. 

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1999; 2:224–233. (c) Rohrer SP, Birzin ET, Mosley RT, Berk SC, Hutchins 

SM, Shen D-M, Xiong Y, Hayes EC, Parmar RM, Foor F, Mitra SW, 

Degrado SJ, Shu M, Klopp JM, Cai S-J, Blake A, Chan WWS, Pasternak A, 

Yang L, Patchett AA, Smith RG, Chapman KT, Schaeffer JM. Rapid 

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through combinatorial chemistry. Science 1998; 282:737–740. (d) Szardenings 

KA, Harris D, Lam S, Shi L, Tien D, Wang Y, Patel DV, Navre M, 

Campbell DA. Rational design and combinatorial evaluation of enzyme 

inhibitor scaffolds: identification of novel inhibitors of matrix metalloproteinases. 

J Med Chem 1998; 41:2194–2200. (e) Kick EK, Roe DC, 

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design and combinatorial chemistry yield low nanomolar inhibitors of 

cathepsin D. Chem Biol 1997; 4:297–307. (f) Combs AP, Kapoor TM, Feng 

S, Chen JK, Daude-Snow LF, Schreiber SL. Protein structure-based 

combinatorial chemistry: discover of non-peptide binding elements to Src 

SH3 domain. J Am Chem Soc 1996; 118:287–288. 

30. Metcal CA III, Eyermann CJ, Bohacek RS, Haraldson C, Varkhedkar VM, 

Lynch B, Bartlett C, Violette S, Sawyer TK. Structure-based design and solidphase 

parallel synthesis of phosphorylated nonpeptides to explore hydrophobic 

binding at the Src SH2 domain. J Comb Chem 2000; 2:305–313. 

31. Such a strategy follows the philosophy of ‘‘resin capture,’’ as introduced by 

Armstrong and Keating: Keating TA, Armstrong RW. Postcondensation 

modifications of Ugi four-component condensation products: 1-isocyanocyclohexane 

as a convertible isocyanide. Mechanism of conversion, 

synthesis of diverse structures, and demonstration of resin capture. J Am 

Chem Soc 1996; 118:2574–2583. See also: Brown SD, Armstrong RW. 

Synthesis of tetrasubstituted ethylenes on solid support via resin capture. 

J Am Chem Soc 1996; 118:6331–6332. 

32. FLO97, Graphics and Molecular Mechanics Software for Drug Design. 

Available from Colin McMartin: cmcma@ix.netcom.com. 

33. McMartin C, Bohacek RS. QXP: powerful, rapid computer algorithms 

for structure-based drug design. J Comput-Aided Mol Design 1997; 11: 

333–344. 

34. The Src SH2 binding site model used in this study was developed based on 

a high resolution (1.0 A ˚ ) crystal structure of Lck SH2 complexed with 

Ac-pTyr-Glu-Glu-Ile-NH 2, obtained from the Brookhaven Protein Data 

Bank (reference code 1LKK). For a description of the X-ray structure, see: 

Tong L, Warren TC, King J, Betageri R, Rose J, Jakes S. Crystal structures 

of the human p56 lck SH2 domain in complex with two short phosphotyrosyl 

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35. Violette SM, Guan W, Bartlett C, Smith JA, Bardelay C, Antoine E, Rickles 

RJ, Mandine E, van Schravendijk MR, Adams SE, Lynch BA, Shakespeare 

WC, Yang M, Jacobsen VA, Takeuchi CS, Macek KJ, Bohacek RS, 


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Src SH2 inhibitors block Src cellular activity and osteoclast-mediated 

resorption. Bone 2001; 28:54–64. 

36. Weigele M, Bohacek R, Jacobsen VA, Macek K, Yang MG, Kawahata NH, 

Sundaramoorthi R, Wang Y, Takeuchi CS, Luke GP, Metcalf CA III, 

Shakespeare WC, Sawyer T. Synthesis of phosphono-containing amino acid 

derivatives and peptides as signal transduction inhibitors. PCT Int Appl 

W099/24442, 1999. 

37. Frost HM, Jee WSS. On the rat model of human osteopenias and 

osteoporosis. Bone Miner 1992; 18:227–236. 

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new, potent, heterocyclic biphosphonate compound. J Bone Miner Res 

1994; 9:745–751. 

39. Violette SM, Shakespeare WC, Bartlett C, Guan W, Smith JA, Rickles RJ, 

Bohacek RS, Holt DA, Baron R, Sawyer TK. A Src SH2 selective binding 

compound inhibits osteoclast-mediated resorption. Chem Biol 2000; 7: 

225–235. 

40. Shakespeare WC, Yang M, Bohacek R, Cerasoli F, Stebbins K, 

Sundaramoorthi R, Azimioara M, Vu C, Pradeepan S, Metcalf CA III, 

Haraldson C, Merry T, Dalgarno D, Narula S, Hatada M, Lu X, van 

Schravendijk M, Adams S, Violette S, Smith J, Guan W, Bartlett C, Herson 

J, Iuliucci J, Weigele M, Sawyer T. Structure-based design of an osteoclastselective, 

nonpeptide Src homology 2 inhibitor with in vivo antiresorptive 

activity. Proc Natl Acad Sci USA 2000; 97:9373–9378. 


3 

Three-Dimensional Structure of 

the Inhibited Catalytic Domain 

of Human Stromelysin-1 by 

Heteronuclear NMR Spectroscopy 

Paul R. Gooley 

University of Melbourne, Parkville, Victoria, Australia 


With the aid of isotopic enrichment it is now routine to determine the 

structure of moderate to large proteins (20 to 40 kDa) by multidimensional 

heteronuclear nuclear magnetic resonance (NMR) spectroscopy [1]. The 

advantages these heteronuclear experiments offer are spectral simplification 

and a reduced dependence on narrow proton linewidths. By spreading 

the 1 H– 1 H correlations of a 2-D NMR spectrum into a third and, perhaps, 

a fourth dimension, according to the chemical shift of the attached 13 Cor 

15 N nucleus, considerable spectral simplification is achieved. As the proton 

of interest is now correlated with its bound 13 Cor 15 N, the information 

content for assignment is increased, and as the individual planes of the 3-D 

or 4-D spectra contain relatively fewer overlapping peaks, problems with 

assignment ambiguities are reduced. These experiments are more efficient 

than their homonuclear counterparts because transfer of magnetization 

relies on the large one-bond heteronuclear couplings (11 to 140 Hz). The 

first stage in solving the structure of a protein requires the acquisition of a 

large number of three-dimensional experiments for sequence-specific as- 


signment of backbone and side-chain atoms. Typically, a dozen or more 2- 

D and 3-D experiments will need to be acquired, taking a total of 4 to 6 

weeks of spectrometer time, thus requiring long-term sample stability or 

readily available large milligram quantities. The second stage in solving a 

structure remains largely dependent on the acquisition of NOE spectra and 

the assignment of many interresidue NOEs. In the special case of a protein 

complex, for example, an inhibited enzyme, where one component is 

isotopically labeled, the spectrum of the complex can be separated into 

subspectra of the two components to aid assignment of both protein and 

ligand resonances and most importantly, to determine protein-ligand 

contacts [2]. Using these methods, the spectrum of the ligand can be solved 

readily providing important information about the conformation of the 

ligand in the bound state. 

The application of these techniques relies on an abundant source of 

13 C- and 15 N-enriched proteins and, therefore, the application of heteronuclear 

NMR spectroscopy to solving the solution structure of proteins 

has relied on advances in molecular biology. Many proteins can be overexpressed 

and isotopically enriched with 13 C and 15 N by replacing the 

carbon source with 13 C-glucose and the nitrogen source with a 15 NH4 + 

salt. Efficient enrichment is possible with media supplemented with minerals 

and vitamins, and using fermentation protocols [3]. Molecular biology 

has further contributed to the number of proteins that can be studied by 

NMR spectroscopy by overexpressing the catalytic or functional domains 

of large proteins, thus truncating the protein to a size (often less than 25 

kDa) that is readily amenable to these techniques [4]. This chapter discusses 

the implementation of these methods to the catalytic domain of 

human stromelysin-1 (sfSTR), a matrix metalloendoproteinase (MMP), 

complexed to a N-carboxylalkyl inhibitor [5–8] (Fig. 1). We will focus on 

the work where the structure of the protein complex was determined and 

compare this structure to other inhibited MMP catalytic domains. 

II. THE MATRIX METALLOPROTEINASE FAMILY 

The matrix metalloendoproteinases (MMPs or matrixins) are a family of 

zinc and calcium dependent extracellular proteases that collectively 

degrade most of the protein constituents of the extracellular matrix [9]. 

There are at least 23 members of this family and are divided primarily on 

the basis of sequence homology and substrate specificity into the 

following grouping: collagenases (MMP-1, -8, -13, -18) gelatinases 


Figure 1 N-carboxylalkyl inhibitor of sfSTR, N-[(R)-carboxyl-ethyl]-(S)-(2phenylethyl) 

glycyl-L-arginine-N-phenylamide [8]. The convention of Schechter 

and Berger [61] is used to describe the specificity subsites of the enzyme S1V, S2V, 

S3V which correspond to the side chains P1V,P2V,P3V of the inhibitor. (From Ref. 5.) 

(MMP-2, -9), stromelysins (MMP-3, -7, -10, -11), membrane-associated 

(MMP-14, -15, -16, -17), and a group of ‘‘others’’ including macrophage 

elastase (MMP-12) and enamelysin (MMP-20). These enzymes participate 

in many normal biological processes such as embryonic development, 

bone remodeling, wound healing, angiogenesis, and apoptosis. 

The pharmaceutical industry has shown considerable attention to the 

regulation of these enzymes because pathological proteolysis by these 

enzymes accompanies many degradative diseases including arthritis, 

ulcerations (corneal, gastric, skin), and periodontal diseases. Degradation 

of the basement membrane by one or more of the MMPs is clearly 

essential in tumor progression. While the activity of the MMPs is 

controlled by endogenous inhibitors such as a-macroglobulin and tissue 

inhibitor of metalloproteases (TIMP-1 and -2), the disease state may be 

a consequence of an imbalance in the ratio of protease to protease inhibitor. 

In the disease state, a potent synthetic inhibitor may have therapeutic 

effects by restoring the ratio of protease to protease inhibitor to normal 

physiological levels. 

The MMPs are synthesized as preproproteins and are secreted as 

latent proproteins. Most MMPs share a common domain structure of a 

propeptide (about 80 amino acids) that has a conserved cysteine ligated to 

the catalytic zinc thus maintaining latency [10], a catalytic domain (about 

180 amino acids), and a C-terminal domain (about 210 amino acids) 


(Fig. 2). Not all MMPs have these domains, for example, matrilysin lacks 

the C-terminal domain and the MT-MMPs do not have the propeptide. 

Some MMPs have additional domains, for example, the gelatinases have 

three repeats of a fibronectin-type II domain. 

The C-terminal hemopexin-like domain has been structurally characterized 

as a four-bladed h-propellor [11–13]. While the structural 

homology of this domain is clear, it has varied functions, for example, it 

is essential for cleavage of triple helical collagens by the collagenases [14]; 

however, it is required for activation of pro-MMP-2 by MT1-MMP [15]. 

The catalytic domain and the C-terminal domain are connected by a 

proline-rich linker that modeling experiments suggest may play a role in 

recognition and destabilization of collagen [16]. 

Figure 2 Domain structure of the MMPs: 92 kDa gelatinase-A (MMP-2), 72 kDa 

gelatinase-B (MMP-9), the collagenases (MMP-1, -8, and –13), stromelysin-1 

(MMP-3) and matrilysin (MMP-7). Matrilysin is the only known MMP that does 

not have a C-terminal hemopexin-like domain. 


The catalytic domain has been the focal point for drug discovery. 

This domain contains the motif HEXXHXXGXXH, which ligates a 

catalytic zinc, and a characteristic h-turn that contains a conserved 

methionine that is structurally located near the catalytic zinc. These 

structural properties have led to the classification of the MMPs with 

several other families of metalloproteases (astacins, serralysins, and reprolysins) 

as ‘‘metzincins’’ [17]. The catalytic domain further contains a 

structural zinc and several calcium ions necessary for stability. The 

catalytic domain of several MMPs has been expressed in bacterial systems 

either as a soluble protein or in inclusion bodies [18, 19]. The proteins have 

been purified and/or refolded and shown to be fully active against small 

peptide substrates. Structures of the catalytic domains have been solved by 

both x-ray crystallography and NMR spectroscopy for collagenase-1, -2 

and -3 [20–25], stromelysin-1 [6,10,26], and matrilysin [27]. Furthermore, 

the pro-form of stromelsyin-1 [10], and the full-length proteins collagenase-1 

[11] and progelatinase A [28] have been solved by x-ray crystallography. 

The structure of the catalytic domains appears identical in the 

truncated forms to that in the full-length protein, and therefore the smaller 

truncated form has been ideal for the structural analysis of the inhibited 

forms to aid inhibitor and drug design. In the following discussion we 

outline the strategy for determining the structure of the inhibited catalytic 

domain of stromelysin-1 by NMR methods. 

III. ASSIGNMENT OF THE RESONANCES 

OF THE INHIBITED CATALYTIC DOMAIN 

OF STROMELYSIN-1 

The assignment of the 1 H, 13 C, and 15 N resonances depends on acquiring 

a large number of separate 3-D or 4-D triple resonance experiments. The 

experiments can be divided into intraresidue and interresidue and, when 

combined, lead to sequence-specific assignment through bonds (Fig. 3) [1]. 

Improvements and new pulse sequences continue; however, a common set 

of experiments to assign the backbone (frequently defined as Ha, Ca, N, 

HN, C’, Ch) resonances of a protein are: 3-D HNCACB, CBCA(CO)NH, 

HCACO, HNCO, (HCA)CO(CA)NH, 4-D HCANNH and HCA(CO) 

NNH [29–32]. Side-chain resonances are assigned using HCCH-TOCSY 

and HCCH-COSY [33]. Unambiguous stereospecific assignment of the 

methyl groups of Leu and Val are possible by preparing a 10% 13 C-labeled 

sample and acquiring a 1 H, 13 C-HSQC spectrum [34]. The incorporation 


Figure 3 3-D and 4-D triple resonance experiments correlate interresidue or 

intraresidue nuclei. (A) The efficiency of the experiments depend on the large 

one-bond couplings; (B) atoms correlated in the 4-D HCANNH, an example of 

an intraresidue experiment; (C) the 4-D HCA(CO)NNH, an interresidue experiment; 

(D) the 3-D HNCO, an interresidue experiment; and (E) the 3-D HCACO, 

an intraresidue experiment. 

of label is nonrandom such that Leu and Val residues are labeled as 

13 Cy2H3- 12 CgH, 13 Cy1H3- 13 CgH, and 13 Cg2H3- 12 ChH, 13 Cg1H3- 13 ChH, 

respectively. Consequently, the 13 Cy2H3 of Leu and 13 Cg2H3 of Val 

groups appear as singlets in the 1 H, 13 C HSQC spectra and are thus readily 

stereoassigned. Measurement of 3 J HNa in 3-D HNHA spectra [35] aids 

determination of f torsion angles and stereoassignment of h-methylene 

groups requires 3-D HNHB [36] and HACAHB [37] experiments. To 

determine the fold of the protein, a large number of interresidue NOEs 

must be assigned in 3-D 15 N-NOESY [38], 3-D and 4-D 13 C-NOESY experiments 

[39,40]. The assignment of the backbone resonances of the 

13 C, 15 N-enriched catalytic domain of stromelysin-1 were mostly accomplished 

with 4-D HCANNH and HCA(CO)NNH experiments (Fig.4) [5]. 

Side-chain atoms were assigned with 3-D HCCH-COSY and HCCH- 

TOCSY experiments with the carrier located near 35 ppm for aliphatic 

side chains and at 124 ppm for aromatic side chains. Stereospecific 

assignment of the methyl groups were obtained with a 10% 13 C-labeled 


Figure 4 Sequential assignment of the backbone atoms for the segment Pro-109 

to Val-113 of inhibited sfSTR by 4-D HCANNH and 4-D HCA(CO)NNH. Four 

planes are shown from each spectrum. The assigned backbone atoms are 

indicated in (A). In (B) the upper four planes in solid lines are from the 4-D 

HCANNH and the lower four planes in dashed lines are from the 4-D 

HCA(CO)NNH. The chemical shifts for the four correlated nuclei in each case 

are shown. The correlations continue for the segment Pro-109 to Pro-129. As Pro 

lacks a protonated N, this residue serves as a ‘‘stop’’ signal. The correlation of 19 

residues with Pro at the N- and C-terminal ends is unique for this segment in the 

sequence of sfSTR, therefore these backbone atoms are specifically assigned 

without having to further assign side chain atoms. (From Ref. 5.) 


sample and a 2-D 1 H, 13 C HSQC spectrum, and couplings from 3-D 

HNHA and HNHB experiments aided defining f and m1 angles. To 

determine which specific ring nitrogens of the six histidine residues ligate 

the catalytic and structural zincs a 2-D 1 H, 15 N HMQC spectrum was 

acquired, where the delay for generating antiphase 1 H, 15 N magnetization 

is set to 22 ms [41]. This experiment will favor the weak two- and threebond 

couplings between the histidine ring nitrogens and the Cy 2H and 

Cq 1H protons (Fig. 5) and clearly showed the Nq2 of His-151, -166, 201, 

-205 and -211, and the Ny1 of His-96 are the zinc ligands. Finally, the 

critical NOEs that describe the tertiary structure of the protein were 

assigned in 3-D 15 N-NOESY, 3D and 4-D 13 C-NOESY experiments. 

IV. ASSIGNMENT OF THE RESONANCES OF THE 

INHIBITOR AND NOEs BETWEEN THE PROTEIN 

AND THE INHIBITOR 

To understand the interactions between a protein and a small ligand, we 

take advantage of the fact that the protein is enriched with 13 Cand 15 N and 

the ligand is not. Pulse sequences can be designed to edit the spectrum of the 

protein-ligand complex into spectra (2-D 1 H, 1 H-COSY, TOCSY, 

NOESY) of the ligand or intermolecular NOEs between the labeled protein 

and unlabeled ligand in either 2-D or preferably 3-D NOESY spectra. 

These experiments are composed of X-half-filters [2,42,43] and either select 

or filter the 13 C, 15 N-attached protons (Fig. 6). Consequently, a 2-D 13 C 

doubly filtered NOESY spectrum will show intramolecular NOEs for the 

ligand, whereas a 3-D 13 C-filtered, 13 C-selected NOESY will show intermolecular 

NOEs between the protein and the ligand. The latter experiment 

is preferably acquired as a 3-D to minimize the ambiguities in assigning the 

protons of the protein that are involved in the interaction with the ligand. 

For the complex of the inhibited catalytic domain of stromelysin-1, 

2-D doubly filtered 1 H, 1 H COSY and TOCSY experiments performed 

poorly. As these experiments depend on 1 H, 1 H couplings, the linewidths of 

the stromelysin-inhibitor complex must be too large for efficient magnetization 

transfer. On the other hand, 2-D doubly filtered NOESY experiments 

acquired in 2 H2O and H2O showed correlations for all protons of 

the inhibitor (Fig. 7), and, as the inhibitor (Fig. 1) was quite simple, the 

resonances were readily assigned. 3-D 13 C-filtered, 13 C-separated NOESY 

experiments were also successfully acquired and assignment of these 

NOEs were unambiguously obtained (Fig. 8). 


Figure 5 Part of a 2-D 1 H, 15 N HMQC spectrum of inhibited stromelysin-1 

where the ring nitrogens of His resonate. The delay where antiphase magnetization 

evolves is set to 22 ms thus favoring the weak two- and three-bond 

couplings of Ny1 and Nq2 to the protons of Cy2H and Cq1H [41]. The 

deprotonated (h-type) nitrogen typically resonates near 249 ppm. These 

resonances for inhibited sfSTR are near 200 to 210 ppm, shifted upfield by 

ligation to the zinc ions. For the stable Ny1-H tautomer two strong couplings are 

observed from the deprotonated Nq2 nucleus to the Cy2H and Cq1H protons. For 

the stable Nq2-H tautomer only one strong coupling is observed from the 

deprotonated Ny1 nucleus to the Cq1H proton. For the imidazolium tautomer the 

resonances of the ring nitrogens are both near 176 ppm and equivalent couplings 

from these nitrogens to both Cy2H and Cq1H protons are observed. For inhibited 

sfSTR, His-151, -166, -201, -205 and -211 are in the Ny1-H tautomer, His-179 is 

in the Nq2-H tautomer and His-96 and -224 are in the imidazolium tautomer. 

Specific labeling of the Ny1 nucleus supports these assignments [5]. (From Ref. 5.) 


Figure 6 X-half filters used for filtering or selecting 13 C and 15 N-attached 

protons. Thick and thin closed rectangles are 180j and 90j pulses, respectively, 

open rectangles are spin lock pulses. (A) A simple X-half filter (2). The delay H is 

equal to (1/(2[ 1 J XH]) where 1 J XH is the one-bond coupling between proton and 

either 13 C (120 to 140 Hz) or 15 N (95 Hz). The second 90j pulse is the editing 


V. STRUCTURE CALCULATIONS 

Peak intensity data from NOE experiments were accumulated and converted 

to interproton distances by calibrating against the expected short 

distances in secondary structure elements. These data were complimented 

with coupling constants determined from the 3-D HNHA and HNHB 

experiments. A total of 2589 peaks were assigned in all NOE experiments. 

After removal of nonconstraining and ambiguous NOEs, typically found 

in mobile regions, 1814 meaningful restraints remained: 325 intraresidue, 

429 sequential, 324 short-range (i+2 to i+5), 665 long range ( > i+5), and 

71 intermolecular. Using a gridsearch program [44] 379 dihedrals (140 f, 

140 c, 99m 1 ) were generated from sequential and intraresidue NOEs and 

coupling constant data from HNHA and HNHB experiments. Structures 

were calculated using the variable target function algorithm DIANA [45], 

but it should be noted that in recent years this method has been replaced by 

torsion angle dynamics methods that are far more efficient [46,47]. To 

determine the structure of the complex, a residue template of the inhibitor 

was built as a single residue covalently linked through an oxygen of the 

carboxylate moeity of the inhibitor (Fig. 1) to the zinc which was 

covalently bonded to the Nq2 of His-201. The residue template of His- 

151 was created with the structural zinc covalently attached to its Nq2 

atom. The structure calculation process is largely iterative with trial structures 

calculated and incompatible NOEs reassigned or removed and new 

NOEs assigned on the basis of agreement with the trial structure. In the 

final calculations, and to reduce bias in structure selection, plots of rmsd 

and number of structures versus target function [48] of the final 80 

pulse. The phase cycling of this pulse with respect to the receiver determines 

whether X-nucleus attached protons are selected or filtered. If both signals are 

added to the receiver (x,x) X-nucleus attached protons are filtered; and if the 

receiver phase is alternated (x,-x) the X-nucleus attached protons are selected. (B) 

A doubly tuned half filter for filtering 13 C attached protons [42]. In this experiment 

the filter consists of two delays (H 1,H 2) tuned to different 1 JCH values 

resulting in superior suppression of artifacts. (C) A doubly tuned time-shared half 

filter for 13 C/ 15 N (43). In this experiment D = 1/(4 1 JNH), D1 = 1/(4 1 JCH) and D2 = 

[1/(4 1 JNH 1/(4 1 JCH)]. Phase cycling the receiver selects or filters both 13 C and 

15 N attached protons. 


Figure 7 2-D 13 C doubly filtered NOESY of inhibited sfSTR using the X-half 

filters of Fig. 4A and B. The NOE correlations of the rings of the P 1V and the P 3V 

group are shown. The Hh and Hg protons of the P1V group were distinguished in a 

similar 2D 13 C doubly filtered TOCSY. The specific assignment of the protons of 

the P 3V group were determined by NOEs between the H2,6 and the NH of the P 3V 

in 2D 13 C, 15 N-filtered experiments using the time-shared doubly tuned half filter 

of Fig. 4C. 


Figure 8 Sections of a 3-D 13 C-separated, 13 C-filtered NOESY of inhibited 

sfSTR. Only NOEs between the 12 C-attached protons of the inhibitor and the 

13 C-attached protons of the protein are observed in this spectrum. These NOEs 

describe the S1V, S2V and S3V subsites of sfSTR. Not shown are several NOEs from 

Val-197 and His-201 to the ethylene group of P 1V. (From Ref. 6.) 

structures were used to select structures for energy minimization using 

the program FANTOM [49]. In the final calculations, 30 structures were 

selected. Table 1 summarizes the DIANA and FANTOM statistics for 

these structures. 

VI. STRUCTURE OF INHIBITED STROMELYSIN-1 

A. The Protein Fold 

Superposition of residues 83 to 248 of the family of structures is shown 

in Figure 9 viewed along the long axis of the catalytic helix. Residues 249 

to 255 are disordered and therefore are not shown. In Figure 10 ribbon 

drawings of two views of the molecule are shown, one from above the 

h-sheet and the other from below the S1’ subsite. The secondary structure 

of sfSTR consists of a five stranded h-sheet with four parallel strands and 


Table 1 Structural Statistics and Residual Violations of the 30 Conformers Used 

to Represent the Solution Structure of the Inhibited Catalytic Domain of 

Stromelysin-1 

Parameter DIANA FANTOM 

DIANA target function (A˚ 2 ) 10.01 F 0.76 

FANTOM energy (kcal/mol) 191.0 F 52.8 

Lennard-Jones energy (kcal/mol) 605.4 F 48.4 

Distance constraint violations (A˚ ) 

sum 35.2 F 1.2 61.6 F 1.4 

maximum 0.48 F 0.06 0.39 F 0.03 

rmsd 0.06 F 0.01 0.08 F 0.01 

Exp. angle constraint violations (j) 

sum 93.4 F 11.1 112.1 F 19.9 

Maximum 7.5 F 0.9 12.6 F 5.0 

Rmsd 0.90 F 0.08 1.2 F 0.3 

Rmsd residues 83–250 (A ˚ ) 

backbone (Ca,N,C’,O) 0.48 F 0.06 0.55 F 0.06 

all heavy atoms 0.94 F 0.06 0.97 F 0.05 


one antiparallel strand and the topology 1x, +2x, +2, 1, using 

the Richardson nomenclature [50]. The h-sheet lies on two helices (helix 

A and B); a third helix (helix C) is near the C-terminus. The molecule 

has two zincs: a catalytic zinc is located at the bottom of a cleft, and 

a structural zinc above the h-sheet. The overall fold of sfSTR may 

be described as follows. The N-terminus is located near the N-terminal 

end of helix C. The protein backbone forms a poorly defined irregular 

strand for the first 13 residues before entering strand I of the h-sheet, 

then descending through helix A. Helix A acts as a backbone to the 

protein, spanning its full length. The pronounced amphipaticity of this 

helix provides hydrophobic residues for internal packing to helix B and 

to the h-sheet, and the hydrophilic residues are exposed to the solvent. 

After helix A the protein backbone turns to form strand II of the h-sheet, 

which lies parallel to and outside strand I. This strand rises steeply, giving 

the h-sheet a distinctly twisted appearance. It is connected by a short 

loop to strand III, which is parallel to and inside of strand I. A long loop 

connects strands III and IV, crossing over strand V and placing strand IV 

along the ligand-binding cleft and antiparallel to strand V. Another small 


Figure 9 Backbone (Ca, C’, N) trace from residues 83 to 250 of 30 conformers 

of inhibited sfSTR. Residues 251 to 255 are disordered and are not included. All 

the heavy atoms of the inhibitor are shown. The family of structures are viewed 

along the long axis of the catalytic helix B. The inhibitor (I) binds to the protein in 

a well-defined cleft and runs antiparallel to the outer strand of the h-sheet with 

the ring of P 1V homophenylalanine (hP) buried in a bottomless S 1V subsite and the 

P2V arginine (R) is exposed to the solvent. 

loop connects strand IV to V, which runs parallel to strand III. The 

structural zinc is ligated by three His, one each from strands IV and V 

(His-166 and -179, respectively), and the third (His-151) from the long 

loop connecting strands III and IV. The fourth ligand of this zinc appears 

to be Asp-153. After strand V the backbone loops to form helix B. The 

two His residues of helix B, His-201 and -205, ligate the catalytic zinc. A 

short turn then enters an extended strand containing His-211, a third 

ligand of the catalytic zinc. From His-211 to Leu-218 several short range 

NOEs, in particular between the side chains of Ser-212 and Ala-217, 

ChH3 of Ala-217 to the NH protons of Leu-218 and Met-219, and the 

backbone atoms of Thr-215 to Ala-217, describes the presence of two 

tight turns. An invariant residue, Met-219, which is residue three in one 

of these turns, is positioned below the three His residues that ligate the 

catalytic zinc and shows NOEs to all three. Except for helix C, the 

remainder of the protein is irregular, but well-defined. Helix C runs 

perpendicular to helix A; the segments C-terminal to these helices are 

near each other. 


Figure 10 Ribbon diagrams of a single conformer of inhibited sfSTR from 

residues 83 to 250. (A) The complex is viewed from above the h-sheet. The 

positions of the two zincs are indicated as large balls. The strands of the hsheet 

(I–V) and helices (A–C) are indicated. The heavy atoms of the inhibitor 

and residues of the protein that ligate zinc are shown. The inhibitor runs 

antiparallel to strand IV. The structural zinc lies above the h-sheet and is 


B. Conformation of the Inhibitor 

The inhibitor binds to the protein in a well-defined cleft (Figs. 9 and 10) and 

in an extended fashion, running antiparallel to strand IV of the sheet, as 

indicated by the strong CaH-CaH NOE between the P2V residue and Val- 

163 (Fig. 8) and parallel to the nonregular loop region encompassing Pro- 

221 to Tyr-223. One of the most striking features of the structure is that the 

S1V subsite appears to pass through the entire structure. Indeed the 

aromatic ring of the homophenylalanine group is clearly observed from 

below the S1V pocket [7]. The S1V subsite is lined with hydrophobic residues 

including Leu-164, Leu-197, His-201, Val-198, Leu-218, Tyr-220, Leu-222 

and Tyr-223. The residues Leu-197, Val-198 and His-201 are from the 

catalytic helix, whereas Tyr-220 and -223 and Leu-218 and -222 are from 

the loop following this helix. Contacts between the protein and inhibitor 

are summarized in Figure 11. Despite the P1V group appearing in contact 

with a number of residues, the ring of this residue can clearly undergo ring 

flips, as indicated by the degeneracy of the H3,5 and H2,6 resonances 

(Fig. 7) thus indicating that this ring is not especially restricted. Similarly 

not all residues of the S 1V are restricted in motion. For example, both 

methyls of Leu-197 show intraresidue NOEs to the CaH proton of Leu-197 

suggesting that motion around the torsion angles m1, m2 is present. We 

note that this residue shows strong NOEs to the homophenylalanine ring 

of the inhibitor (Fig. 8) indicating that it is in contact with the inhibitor. 

Analysis of spectra with other inhibitors with extensions to the homophenylalanine 

showed this residue became restricted in motion, and thus 

subtle changes to residue mobility is inhibitor dependent. 

The family of conformers were analyzed for hydrogen bonds, where 

acceptor-donor (N-H...O) distance was set to an upper limit of 2.4 A ˚ and 

ligated by His-166 from strand IV, His-179 from strand V, and His-151 and 

Asp-153 both from a 14 residue loop. The catalytic zinc is ligated by His-201 

and –205 from helix B and His-211. (B) The complex is viewed from below S1V 

subsite. The heavy atoms of the inhibitor and the residues that are in 

intermolecular contact (Leu-164, Leu-197, Val-198, His-201, Leu-218, Tyr-220, 

Leu-222, Tyr-223) with the P1V homophenylalanine are shown. To reduce 

crowding in the figure not all these residues are labelled. (*) marks Leu-218 

and His-201. Val-198 is below Leu-197. Leu-164 is at the N-terminal end of 

the h-strand that appears above Leu-197 in this figure. The ribbon diagrams 

were produced by MOLSCRIPT [62]. 


Figure 11 Potential hydrogen bond partners to the backbone atoms of the 

inhibitor and the residues of the S1V subsite that are in intermolecular contact with 

the P1V homophenylalanine. 

the angle to 35j. The analysis suggests that the NH of the P3V hydrogen 

bonds to the carbonyl of Asn-162; the carbonyl of P1V hydrogen bonds to 

the NH of the Leu-164 (which is slowly exchanging with deuterium); and 

the amine of P 1V hydrogen bonds with the carbonyl of Ala-165. The 

structures described here do not show hydrogen bonds between the NH 

and the carbonyl of the P2V arginine to the protein, which is in contrast to 

reported crystal structures which show a hydrogen bond to the NH of Tyr- 

223 [10]. Although Pro-221 and Tyr-223 are near atoms of the inhibitor, for 

example, the NH of Tyr-223 shows weak NOEs to the ring of P3V, their 

distances in the structure models are not in agreement with these residues 

participating in hydrogen bonds. The NH of Tyr-223 does not show slow 

exchange with 2 H 2O and analysis of 2-D saturation transfer difference 

1 H, 15 N HSQC spectra suggested that the exchange rate of the NH of Tyr- 

223 was one to two orders slower than a free amide proton further 


supporting the lack of a strong hydrogen bond [7]. The modest protection 

of the amide from solvent exchange may simply be due to solvent 

accessibility. We also note that the conditions of data collection for the 

NMR solution structure were 40jC, which may destablize any weak 

hydrogen bonds observed in the crystal structure. 

VII. COMPARISON OF INHIBITED STROMELYSIN 

TO OTHER MMPS 

The structures of the catalytic domain of a large number of MMPs have 

been solved by x-ray crystallography and NMR spectroscopy [7,10,20– 

27]. All cases show that this catalytic domain of the MMPs has a common 

fold to that described above, suggesting that the design of specific 

inhibitors will require detailed structural investigations that take advantage 

of differences of the specificity pockets. The most significant difference 

to date has been the nature of the S1V subsite, which is clearly very deep in 

stromelysin and collagenase-3 [7,10,25], deep in collagenase-2 [21], to quite 

shallow for collagenase-1 [20,23,25] and matrilysin [27]. In collagenase-1 

an arginine residue (equivalent to Leu-197 in stromelysin-1) hydrogen 

bonds to a structural water and delimits the S1V subsite. Consequently, 

many inhibitors with large bulky P1V groups show poor affinity for 

collagenase-1. However, it has been observed that this protein can undergo 

a conformational change to accommodate such groups [25]. The catalytic 

domain of stromelysin-1 has been studied by NMR spectroscopy as a 

complex with a variety of inhibitors [7,10,51], with most binding with 

groups in the S1V to S3V subsites. Those with a thiadizole group ligating the 

catalytic zinc bind with groups in the nonprime (S1 to S3) subsites. These 

inhibitors show NOEs and thus contacts to residues in the S3 subsite 

including His-166, Tyr-155, and Tyr-168, which are located near the 

structural zinc. 

An advantage of NMR spectroscopy is the analysis of protein 

dynamics. Measurement and analysis of the relaxation parameters R1, 

R2, and the 15 N NOE of 15 N-labeled proteins leads to an order parameter 

(S 2 ) that can describe the relative mobility of the backbone of the protein. 

Both collagenase-1 and stromelysin-1 have been studied either as inhibited 

complexes or the free protein [19, 52]. Stromleysin-1 was studied with 

inhibitors binding to prime or nonprime subsites. Presence or absence of 

inhibitors in the nonprime sites had minor effects on the highly ordered 

structure of residues in these subsites, which are in contact with the 


inhibitor. Inhibitors binding to the primed subsites induced considerable 

order in the regions 191 to 192 and 223 to 224. Most importantly, the 

amide proton of His-223 formed a hydrogen bond to a carbonyl group of 

the inhibitor. In addition to these changes residues remote from the 

inhibitor, but near the binding sites showed increased mobility. These 

results suggest that the rigidity of the S1 to S3 subsites are important for 

distinguishing between ligands, while the flexible S 1V to S 3V subsites are 

more accommodating to a broad range of residues. The flexibility of the 

S1V subsite is in agreement with our observations. Interestingly, similar 

studies on collagenase-1 with a hydroxymate inhibitor [19] bound to the 

S1V to S3V subsites showed the analogous region to 220-226 of stromelysin- 

1 was disordered in both the presence or absence of inhibitor. All these 

results indicate that changes to mobility are complex and mostly unpre- 

Figure 12 Catalytic mechanism of thermolysin and stromelysin-1. (A) The 

mechanism of thermolysin [54]. (B) The mechanism of stromleysin-1 [10]. 

Equivalent residues to Tyr-157 and His-231 are not observed for stromelysin-1. 

The proposed mechanism for collagenase-1 [53] is similar to stromelysin-1, but 

also involves Asn-180 (equivalent to Asn-162 in stromelysin-1). This residue 

cannot participate in stromelysin-1 due to an additional residue between Ala-165 

and Asn-162. (Adapted from Ref. 10.) 


dictable: however, such analyses may prove useful in supporting and 

monitoring the presence of stabilizing interactions. 

The mechanism proposed for fibroblast collagenase [53] and stromelysin-1 

(10) is similar to that suggested for thermolysin [54] (Fig. 12). In 

thermolysin the zinc ion ligates the carbonyl of the substrate and with Tyr- 

157 and His-231 stabilizes the tetrahedral intermediate. In collagenase-1 

and stromelysin-1, however, the stabilization of the carbonyl of the 

substrate and the tetrahedal intermediate is by zinc alone. In thermolysin 

the NH of the scissile bond is stabilized by a peptide carbonyl of Ala-182 

and the side chain carbonyl of Asn-112. For collagenase-1 similar interactions 

by the peptide carbonyl of Ala-182 and the carbonyl of the side 

chain of Asn-180 are suggested. For stromelysin-1, however, only the 

carbonyl of Ala-165 would be involved in the stabilization of the NH of 

the substrate; the equivalent Asn (Asn-162) is not involved as there is a 

residue insertion in the stromelysin-1 sequence compared with the 

collagenase-1 sequence. The proposed mechanisms of thermolysin, collagenase-1 

and stromelysin-1 suggest that the Glu in the consensus 

sequence HEXXH would promote the nucleophilic attack of water on 

the scissile bond of the peptide substrate. The solution structure of 

stromelysin-1 described here lacks the rigidity expected for the side chain 

of this residue, Glu-202. In several members of the family of structures, 

however, this side chain does approach a position that is consistent with 

the mechanistic role of this residue. 

VIII. CONCLUSION 

This chapter has discussed the use of heteronuclear NMR and isotope 

editing methods to determine the structure of protein complexes of 

therapeutically important drug targets. NMR methodology continues 

to develop with larger protein complexes being studied, and more 

accurate structures being determined. Developments include deuteration 

of proteins [1] to enhance relaxation properties, and experiment design, 

for example, Transverse Relaxation Optimized Spectroscopy (TROSY) 

[55], which takes advantage of favorable relaxation pathways thus 

allowing proteins of at least 60 kDa to be studied; inclusion of residual 

dipolar couplings as an orientation constraint in structure calculations 

[56,57] are increasing the accuracy of solution structures; and combining 

deuteration and TROSY experiments has allowed hydrogen bonds to be 

directly observed and also included in structure calculations [58]. An 


additional and powerful application of NMR spectroscopy is the method 

of ‘‘NMR by SAR’’ developed by Fesik et al. [59], which has been applied 

to finding new drug leads for stromelysin-1 [60]. NMR spectroscopy has 

clearly become a powerful and essential tool in the design and development 

of novel drug leads. 

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4 

Cannabinergics: Old and New 

Therapeutic Possibilities 


University of Connecticut, Storrs, Connecticut, U.S.A. 

Andreas Goutopoulos 

Serono Reproductive Biology Institute, Rockland, Massachusetts, U.S.A. 


Cannabis sativa, one of the oldest plants farmed by man, has been known 

for its medicinal properties for at least four millennia (Peters, 1999). The 

psychoactive–euphoric effects of this plant, as well as its facile and wide 

climatic range of cultivation, have rendered it a very popular recreational 

drug. Today, cannabis, or marijuana, is still the focus of strong social, 

legal, and medical controversy over its therapeutic utility. 

Referenda in Arizona and California in 1997, and later, others in 

eight additional states, aimed at legalizing marijuana cigarettes for medical 

purposes. Two licensed, single-compound, cannabimimetic pharmaceuticals, 

Marinol (Dronabinol, delta-9-THC from Roxane Labs) and Cesamet 

(Nabilone, developed at Eli Lilly, now in use in the United Kingdom), are 

marketed for two purposes: to control the nausea and emesis produced by 

cancer chemotherapy and to serve as appetite stimulants in AIDS-related 

anorexia. In clinical trials with cancer chemotherapy patients, both these 

agents have proven to be superior to conventional antiemetics, such as 

perchlorperazine (Breivogel, 1998). 

Beyond this relatively limited medical use of cannabimimetics, the 

current, albeit long-delayed elucidation of their pharmacology is likely to 

lead to a wide expansion of the clinical potential and significance of 


these drugs. The oily, noncrystalline nature of the biologically active 

terpenoid ingredients of Cannabis sativa contributed to the lag in understanding 

of cannabinoid biology. The main active ingredient, (–)-delta-9tetrahydrocannabinol 

(delta-9-THC), was isolated and identified only in 

1964 (Mechoulam, 1967), over a hundred years after the isolation of many 

important crystalline biologically active natural products, such as morphine 

and quinine. A second reason for the lack of progress in defining the 

biology of cannabimimetics was the long-standing scientific misconception 

that the cannabinoid-induced pharmacological actions are mediated by 

perturbation of cellular membranes rather than through specific receptors. 

This hypothesis was a deterrent in the pursuit of possible specific cannabinoid 

binding sites. Owing to their high lipophilicity, cannabinoids were 

paralleled with general anesthetics in terms of their mechanism of action 

(Paton, 1975). Although cannabinoids were found to clearly perturb membranes 

(Makriyannis, 1987), such effects were never proven to be directly 

responsible for their biological activity. 

The advent of synthetic cannabimimetics with a high degree of 

enantioselectivity (Johnson, 1986; Little, 1988) paved the road for the 

identification of specific cannabinoid binding sites in rat brain (Devane, 

1988). This discovery marked the onset of a revolution in the understanding 

of cannabinoid biology. 

II. CANNABINOID RECEPTORS 

A. The CB1 Receptor 

Definitive proof of the existence of the cannabinoid receptor came with 

the isolation of the cDNA of a cannabinoid receptor from a rat cerebral 

cortex cDNA library and its expression in Chinese hamster ovary 

(CHO) cells (Matsuda, 1990). A year later, the corresponding human 

receptor, named CB1, was cloned and found to share a 97.3% homology 

with the rat receptor (Gerard, 1991). The CB1 472 amino acid 

sequence revealed (Matsuda, 1990; Gerard, 1991) that it is a member 

of the G-protein-coupled receptors (GPCRs). Receptors of this family 

are membrane embedded and consist of an extracellular N-terminus, 

seven transmembrane helices interconnected with intra- and extracellular 

loops, and an intracellular C-terminus. Bramblett et. al. (1995) constructed 

a model for CB1, using the known structure of bacteriorhodopsin 

as a starting point. 


The sites involved in interactions with G proteins of the Gi/o family 

are the third intracellular loop from the N-terminal side and the Cterminus 

(Howlett, 1998a). The C-terminus was found to bind with high 

affinity to Gi and the synthetic C-terminus peptide was found to individually 

stimulate GTPgS binding to G protein and to inhibit adenylate 

cyclase (Howlett, 1998b). Similarly with other GPCRs, CB1 is allosterically 

regulated by sodium ions. It has been shown that sodium ions affect 

both ligand binding and signal transduction by inducing a receptor 

conformational change (Houston, 1998). There is also evidence that an 

interhelical H-bonding interaction between helix II Asp and helix VII Asn 

is important for the stabilization of a receptor conformational state that 

has high affinity for most cannabimimetic ligands (Tao, 1998), (Howlett, 

1998a). Sodium ions presumably disrupt this H bond, and thus, result in a 

different, low affinity, receptor state. 

The CB1 receptor is coupled with G i (Howlett, 1998a). CB1 

activation leads to inhibition of adenylyl cyclase and, therefore, to reduction 

of cAMP levels. Many eukaryotic cells utilize cAMP as a 

second messenger that activates the cAMP-dependent protein kinase A 

(PKA), which in turn phosphorylates various proteins, regulating their 

function. One of the cAMP-dependent cannabinoid effects is the 

enhancement of voltage-sensitive, outwardly rectifying potassium channels, 

which occurs as a result of decreased phosphorylation of the K + 

channel protein by PKA (Deadwyler, 1995). Besides G i, CB1 is coupled 

to G o (Howlett, 1999). Furthermore, apart from inhibition of adenylyl 

cyclase, CB1 utilizes several additional effector systems (intracellular 

mediators) involving Gi/o proteins: the inhibition of N-type Ca 2+ channels 

(Mackie, 1992); the activation of mitogen-activated protein kinase 

(MAP kinase) (Bouabula, 1995a); and the expression of immediate early 

genes like Krox-24 (Bouabula, 1995b). Other cannabinoid-induced cellular 

effects include activation of inwardly-rectifying potassium channels 

(Pertwee, 1997) and possibly the activation of phospholipases A, C, or D 

(Felder, 1995). 

Different G proteins or second messengers may be coupled to CB1 

in different brain regions and may mediate different physiological effects 

(Howlett, 1999). Utilization of diverse effector systems by CB1 may 

explain how the response to cannabimimetics varies across different cell 

types. Understanding which physiological responses are mediated by 

each of the foregoing intracellular signaling systems is of great significance 

and may suggest new approaches for the design of selective 

cannabimimetic agents. 


B. The CB2 Receptor 

Homology cloning revealed the existence of a second cannabinoid receptor, 

CB2 (Munro, 1993). This receptor shows 44% homology to the total 

CB1 and 68% homology within the transmembrane regions. Not present 

in the brain in significant levels, CB2 is found mainly in the periphery and 

particularly in tissues of the immune system, such as leukocytes, spleen, 

thymus, and tonsils (CB1 is found in some of these cells as well). Localization 

of CB2 in the immune system suggests an immunomodulatory role 

for this receptor. Thus, CB2 is likely to be the mediator of the long-known 

immunosuppressive properties of marijuana. Similarly to CB1, CB2 uses 

signal transduction pathways, such as inhibition of adenylyl cyclase and 

stimulation of MAP kinase. However, unlike CB1, CB2 does not affect ion 

channels (Pertwee, 1997). 

Although the human genome does not contain genes with high 

homology to those of CB1 and CB2, other cannabinoid receptor types 

may exist nevertheless. An amino-terminal differentially spliced CB1 

variant, CB1A, has been isolated from a human lung cDNA library and, 

akin to CB1, is expressed in the brain (Shire, 1995). The biological role and 

pharmacological implications of this variant are still unclear. 

The existence of a second peripheral CB2-like receptor is supported 

by the finding that palmitylethanolamide provided antinociception after 

intraplantar injection of formalin solution in mice paws (Calignano, 1998). 

This effect was attenuated by a CB2 antagonist, SR144528, but not by 

SR14176A (a CB1 antagonist) nor by the opioid antagonist naloxone. 

Palmitylethanolamide has no significant affinity for either CB1 or CB2 

(Khanolkar, 1996). However, in addition to the findings of Calignano 

et al., palmitylethanolamide is shown to have a down-regulating effect 

on mast cell activity, presumably mediated through a CB2-like receptor 

present in these cells (Facci, 1995). 

Mouse vas deferens (MVD) seems to express CB1 and at least one 

CB2-like cannabinoid receptor type, as is demonstrated by the presence of 

CB1 and CB2-like mRNA as well as by data collected from experiments 

with cannabinoid receptor selective agonists and antagonists (Pertwee, 

1999). Furthermore, evidence indicates that a CB1-like receptor exists in 

vascular endothelium, which upon activation produces significant hypotension 

(Wagner, 1999). This receptor differs from CB1 in its pharmacological 

response to some well-characterized cannabimimetics. 

None of these possible new CB variant receptors has been cloned 

yet; therefore, their existence is still putative. Thus, it is unclear whether 


these observations are indicative of novel cannabinoid receptor types, 

results of alternative versions of known receptors coupled with different 

effector systems, or even results of different affinity states of a single 

cannabinoid receptor. Discovery and characterization of new cannabinoid 

receptors with different distribution patterns and ligand affinities is of 

major importance because it will provide new targets for the development 

of highly selective and clinically useful cannabinergic agents. 

C. Cannabinoid Receptor Distribution 

The ubiquitous CB1 is found in the central nervous system (CNS), as 

well as in the periphery and in both neural and nonneural tissues; it 

is one of the most abundant GPCRs in the brain (Breivogel, 1998). 

As shown by autoradiographic studies in various mammalian brains 

(Herkenham, 1990; Gatley, 1998), CB1 density is highest in basal ganglia: 

substantia nigra pars reticulata, entopeduncular nucleus, and the 

external segment of globus pallidus. Moderately high CB1 density is 

found in putamen, cerebellum, and hippocampus, whereas moderate 

levels exist in cerebral cortex. The spinal cord shows a range of moderate 

densities, while thalamus and brain stem contain low to negligible levels. 

Autoradiography studies with [ 35 S]GTPgS revealed that cannabinoid 

activity occurs with the same regional distribution as the receptors; 

however, the level of activity did not parallel receptor density (Breivogel, 

1998). This pattern of CB1 distribution in the brain is similar to that of 

D1 receptors, which suggests that the cannabinoid system may be 

involved in the modulation of the dopaminergic activity (Gatley, 1998). 

In fact, CB1 mediates a negative feedback control over D 2 in the striatum 

(Giuffrida, 1999). 

In the periphery, CB1 is found in the adrenal glands, bone 

marrow, heart, lung, prostate, testes, thymus, tonsils, spleen, lymphocytes, 

phagocytes, smooth muscle, vascular endothelium, peripheral 

neurons (e.g., in the gut), kidneys, uterus, and sperm as reviewed by 

Schuel et al. (1999). 

The CB2 receptor has a more limited distribution, being localized 

predominantly in the immune system. Among the human leukocytes, B 

lymphocytes express the highest levels of CB2, followed respectively by 

natural killer cells, monocytes, polymorphonuclear neutrophils, T8 lymphocytes, 

and T4 lymphocytes. It is also found in the lymph nodes, spleen, 

tonsils, and thymus (Cabral, 1999). 


D. Cannabinoids and Membranes 

Before the first indication of the existence of cannabinoid receptors, the 

prevailing theory on the mechanism of cannabinoid activity was that 

cannabinoids exert their effects by nonspecific interactions with cell 

membrane lipids (Makriyannis, 1990). Such interactions can increase the 

membrane fluidity, perturb the lipid bilayer and concomitantly alter the 

function of membrane-associated proteins (Loh, 1980). A plethora of 

experimental evidence suggests that cannabinoids interact with membrane 

lipids and modify the properties of membranes. However, the relevance of 

these phenomena to biological activities is still only, at best, correlative. An 

important conundrum associated with the membrane theories of cannabinoid 

activity is uncertainty over whether cannabinoids can achieve in 

vivo membrane concentrations comparable to the relatively high concentrations 

used in in vitro biophysical studies (Makriyannis, 1995). It may be 

possible that local high concentrations are attainable under certain physiological 

circumstances, and, if so, some of the cannabinoid activities may 

indeed be mediated through membrane perturbation. 

Interactions of cannabimimetics with membranes may be of importance 

for auxiliary roles such as transport to their sites of action and proper 

orientation for optimum interaction with their receptors. The molecular 

features of cannabimimetics are shown to govern the manner by which 

these molecules cross cell membranes, including the brain–blood barrier 

(BBB) (Makriyannis, 1995). Most cannabimimetics are amphipathic, a 

property that affects their orientation within the lipid bilayer. Strong 

experimental evidence has shown that the phenolic hydroxyl group of 

(–)-delta-9-THC anchors it at the polar interface of the membrane, 

whereas the tricyclic hydrophobic system remains imbedded in the bilayer 

and perpendicular to the fatty acid chains (Martel, 1993; Makriyannis, 

1995). Other active dihydroxy THC derivatives adopt similar orientation, 

while many inactive analogs assume an orientation parallel to the lipids 

and a position deeper, closer to the center of the bilayer. The proper 

positioning and orientation of cannabinoids within the membrane may be 

crucial for reaching the receptor site, located within the transmembrane 

receptor helices, by lateral diffusion (Makriyannis, 1995). 

III. THE ENDOGENOUS LIGANDS 

The discovery of the cannabinoid receptors and their G-protein-coupled 

nature strongly suggested the existence of endogenous cannabimimetic 


Figure 1 The structure of anandamide. 

ligand(s) able to exert physiological activity upon binding to these receptors. 

Initial efforts to identify a possible protein (Nye, 1988) or a watersoluble 

endogenous cannabimimetic ligand were unsuccessful (Deadwyler, 

1995). The hypothesis that such a putative endocannabinoid should be 

lipophilic, like the classical exogenous cannabinoids, led Mechoulam et al. 

to seek such a ligand in the hydrophobic fractions of porcine brain extracts 

(Devane, 1992). Repetitive fractionations and purifications led to the 

identification of a substance that bound to CB1 in a saturable fashion. 

This compound was the ethanolamide of arachidonic acid (arachidonyl 

ethanolamide, AEA) (Fig. 1). The authors named this brain constituent 

anandamide from ananda, the Sanskrit word for bliss. 

Anandamide is found in human brain: 100 pmol/g in the hippocampus, 

75 pmol/g in the thalamus, 60 pmol/g in the cerebellum, and 55 

pmol/g in the striatum (Martin, 1999). The concentration of AEA increases 

postmortem, especially when the brain is kept at ambient temperature. 

Furthermore, AEA surges are observed when cerebellar granule cells are 

treated in hypoxic conditions (Hillard, 1997). Although such concentration 

increases may be artifacts of postmortem brain damage, they may also 

occur in living tissue under certain conditions, such as hypoxia. 

Outside the CNS, anandamide is found in the spleen and heart at 

approximately 10 pmol/g (Martin, 1999). It is also localized in rat testes 

and uterus in concentrations significantly greater than those in the brain 

Figure 2 The structures of two N-acylethanolamide (NAE) endocannabinoids. 


Figure 3 The structure of 2-arachidonyl glycerol. 

(Schmid, 1997). Very low levels have been detected in serum, plasma, and 

cerebrospinal fluid—a fact that indicates that anandamide is not hormonal 

in nature but rather is biosynthesized at or near its sites of action. 

In addition to anandamide, several other endogenous polyunsaturated 

fatty acid derivatives were also found to act as cannabimimetics. 

They are all now collectively referred to as endocannabinoids. Soon after 

the discovery of anandamide, two more fatty acid ethanolamides were 

isolated and found to bind to CB1 preparations with affinities similar to 

that of anandamide (anandamide CB1 binding affinity Ki = 39.2 nM, 

according to Hanus et al., 1993). These were the homo-g-linolenylethanolamide 

(CB1 Ki = 53.4 nM) and 7,10,13,16-docosatetraenylethanolamide 

(CB1 Ki = 34.4 nM) (Fig. 2). All three N-acylethanolamide endocannabinoids 

were found to be CB1 agonists in the MVD test (Pertwee, 1994). 

A different type of endocannabinoid that is also an arachidonic acid 

derivative was first isolated from canine gut and identified as 2-arachidonyl 

glycerol (2-AG) (Fig. 3) (Mechoulam, 1995a). 

Later 2-AG was also found in the brain (Stella, 1997) and spleen 

(Di Marzo, 1998). It was shown to be released in a calcium-dependent 

manner, reaching concentrations 170 times higher than that of anandamide 

in the brain (Stella, 1997). Like the other endocannabinoids, 2-AG 

was shown to produce the typical tetrad of cannabimimetic behavioral 

effects and inhibit electrically evoked contractions of mouse MVD 

(Mechoulam, 1995a). 

A. Anandamide Pharmacology 

Since the discovery of anandamide in 1992, a number of studies have 

examined its pharmacological properties. Although its roles are still 

elusive, a plethora of data supports the initial postulate that anandamide 

is the major endogenous cannabinoid ligand. As mentioned earlier, anandamide 

binds to CB1 from brain preparations and displaces various well- 


characterized cannabimimetic radioligands (Hillard, 1997). Furthermore, 

it binds to CB1 expressed in cells transfected with CB1 DNA (Vogel, 1993). 

Its CB1 affinity is comparable to that of delta-9-THC. Anandamide does 

not have effects on other than the cannabinoid receptors (Hillard, 1997) 

and has cannabimimetic properties, both in vitro and in vivo. Anandamide 

acts as a CB1 agonist, as demonstrated when it inhibited forskolinstimulated 

adenylyl cyclase activity in N18TG2 cells (IC 50 = 540 nM) 

(Vogel, 1993), CB1 expressing CHO cells (IC 50 = 160–322 nM) (Vogel, 

1993), and cerebral membranes (IC50 =1.9AM) (Childers, 1994). It was 

found to have lower efficacy (lower maximal effect) than the high-affinity 

cannabimimetics WIN55212-2 and CP-55,940; thus, anandamide was classified 

as a partial agonist (Vogel, 1993; Childers, 1994). Furthermore, it was 

found to have inhibitory effects in N-type calcium currents through a pertussis-toxin-sensitive 

pathway in N18 neuroblastoma cells (Mackie, 1993). 

Anandamide, in vivo, was shown to produce the four characteristic 

effects of cannabimimetics, namely, analgesia, hypothermia, hypoactivity, 

and catalepsy (Smith, 1994; Fride, 1993; Crawley, 1993). These four 

effects are not unique to cannabimimetics; when they are produced 

together, however, they are highly predictive of cannabimimetic activity 

(Martin, 1991). Anandamide was found to be less potent than delta-9- 

THC in producing these behavioral effects in mice (Fride, 1993). It has 

quicker onset and shorter duration of action, the latter because of rapid 

catabolism. Cross-tolerance studies, in which pretreatment of mice with 

delta-9-THC produced tolerance to most of the pharmacological effects 

of anandamide and vice versa, indicate that both drugs act on the same 

receptor (Jarbe, 1998). 

In addition, anandamide was found to parallel classical cannabinoid 

pharmacology in a series of nonbehavioral experimental systems. In 

isolated MVD, (Pertwee, 1992) and guinea pig ileum, it inhibited electrically 

evoked twitch responses (Pertwee, 1995). Moreover, anandamide was 

shown to decrease intraocular pressure in rabbits (Pate, 1995), to reduce 

sperm-fertilizing capacity in sea urchins by inhibition of the acrosome 

reaction (Schuel, 1994), and to produce hypotension in rats (Varga, 1995). 

All the foregoing pharmacological effects of anandamide, in conjunction 

with the well-documented existence of specific systems for its 

biosynthesis, catabolism, and cellular reuptake to be discussed shortly, 

suggest that anandamide is indeed the endogenous cannabinoid ligand. 

The other two less studied N-acylethanolamide endocannabinoids and also 

2-AG may serve similar functions. The differential roles of each of these 

four endocannabinoids are still unclear. 


Despite all the research conducted on the pharmacological effects of 

anandamide and other cannabimimetics, the exact role of the endogenous 

cannabinoid system remains elusive. The ubiquitous pharmacology of 

cannabimimetics suggests that endocannabinoids have differing functions 

depending on the tissue or organ system. A recent report (Giuffrida, 1999) 

sheds light on the role of anandamide in the dorsal striatum. It was shown 

that anandamide, but not 2-AG, is released after D 2 activation and 

subsequently suppresses motor activity, possibly by inhibiting postsynaptic 

GABAergic currents. Therefore, it was suggested that anandamide, at 

least in the striatum, plays the role of an autocoid (local neuromodulator) 

that has a negative feedforward regulatory effect of D2-mediated locomotor 

behavior (Giuffrida, 1999). 

It may be that 2-AG has different roles in the CNS, for it can reach 

170 times higher concentrations than that of anandamide in the brain 

(Stella, 1997), even though it was undetectable in the striatum. In the 

hippocampus, 2-AG, but not anandamide, was released after glutamatergic 

activation (Stella, 1997). Sugiura (1999) found 2-AG to be a full CB1 

agonist, whereas anandamide is a partial agonist, again pointing to 

alternative roles for 2-AG in comparison to anandamide. 

B. Endocannabinoid Metabolism 

Biosynthesis of Anandamide 

Considerable advances have been made during the late 1990s toward 

understanding the physiological pathways that are involved in the synthesis 

and inactivation of endocannabinoids. The first of these pathways to 

be observed, an enzymatic activity responsible for anandamide hydrolysis, 

led to lower apparent CB1 affinities for anandamide analogs in studies 

involving structure–activity relationships (SARs) (Childers, 1994), 

(Abadji, 1994). Inclusion in the binding assay of phenylmethanesulfonyl 

fluoride (PMSF), a general serine protease inhibitor, protected the anandamide 

analog from hydrolysis (Abadji, 1994; Khanolkar, 1996). Shortly 

after, an enzyme specific for this hydrolytic process was identified and 

characterized (Deutsch, 1993; Ueda, 1995). Initially, it was thought that 

this hydrolase, named anandamide amidase or fatty acid amidohydrolase 

(FAAH), was also responsible for the synthesis of anandamide by acting 

reversibly (Devane, 1994). However, the current belief is that anandamide 

amidase is unlikely to be physiologically responsible for anandamide 

synthesis because of the requirement for significantly higher than normal 


physiological concentrations of arachidonic acid and ethanolamine (up to 

160 mM) for this enzyme to catalyze the reverse reaction (Piomelli, 1998). 

Therefore, it is currently believed that anandamide is formed from 

membrane phospholipids (Fig. 4) through a pathway that involves: (1) a 

trans-acylation of the amino group of phosphatidylethanolamine with 

arachidonate from the sn-1 position of phosphatidylcholine and (2) a Dtype 

phosphodiesterase activity on the resulting N-arachidonylphosphatidylethanolamide 

(NAPE). Synthesis of anandamide is presumably regulated 

at the levels of both enzymes, the N-acyltranferase and the 

phospholipase D, by stimuli that raise intracellular calcium or by receptors 

linked with cAMP and PKA. It has been shown that anandamide is formed 

when neurons are depolarized and, therefore, the intracellular calcium ion 

levels are elevated (Cadas, 1996). 

Biosynthesis of 2-AG 

Two possible pathways for the biosynthesis of 2-AG have been proposed: 

(1) a phospholipase C (PLC) hydrolysis of membrane phospholipids 

followed by a second hydrolysis of the resulting 1,2-diacylglycerol by 

diacylglycerol lipase or (2) a phospholipase A1 (PLA1) activity that 

generates a lysophospholipid, which in turn is hydrolyzed to 2-AG by 

lysophospholipase C (Fig. 5) (Piomelli, 1998). Alternative pathways may 

also exist from either triacylglycerols by a neutral lipase activity or 

lysophosphatidic acid by a dephosphorylase. The fact that PLC and 

diacylglycerol lipase inhibitors inhibit 2-AG formation in cortical neurons 

supports the contention that 2-AG is, at least predominantly, biosynthesized 

by the PLC pathway (Stella, 1997). However, a mixed pathway may 

also be plausible. 

As with the biosynthesis of anandamide, the biosynthesis of 2-AG is 

also triggered by increases of intracellular calcium ions that result from 

neuronal activity. High frequency stimulation of neurons produced a 

fourfold increase of 2-AG accumulation compared with controls, and this 

was prevented by sodium ion channel blocking or removal of calcium ions 

(Stella, 1997). The concentration of 2-AG in depolarized neurons reached 1 

to 2 AM, significantly higher than anandamide and sufficient to substantially 

activate CB1 (Stella, 1997). 

Based on the pathways just proposed for the biosynthesis of anandamide 

and 2-AG, the formation of these endogenous ligands must also be 

dependent on the composition of the precursor lipids. This dependence is 

of greater importance for anandamide rather than for 2-AG because 


Figure 4 Anandamide biosynthesis. 


Figure 5 Possible 2-AG biosynthesis. 

arachidonic acid is rarely esterified at the sn-1 position of phospholipids, 

whereas it is commonly found at the sn-2 position. 

Endocannabinoid Release 

Immediately after synthesis, endocannabinoids are released in the extracellular 

space, where they then act on the same or neighboring cells as 

autocrine or paracrine mediators (Di Marzo, 1999). Experimental evidence 

thus far indicates that anandamide and 2-AG, unlike other classical 

neurotransmitters, are not stored in vesicles. First, anandamide basal 

concentrations are extremely low (5–10 pmol/g), 100 to 10,000 times lower 

than those of classical neurotransmitters (Cadas, 1997). Second, stimulus-dependent 

anandamide release is linked with de novo NAPE and 


subsequent anandamide biosynthesis (Cadas, 1996; Di Marzo, 1999). 

Therefore, it is currently believed that anandamide and 2-AG are produced 

and immediately released from the neurons upon demand (Di Marzo, 

1999b; Piomelli, 1998). 

The poor water solubility of anandamide must preclude extensive 

free diffusion in the extracellular space. Since, however, anandamide is 

found in brain incubation media or perfusates of brain microdialysis 

experiments it obviously exits the cells (Giuffrida, 1999). Additionally, it 

is known that striatal astrocytes, which do not produce anandamide, do 

respond to it (Cadas, 1996). Therefore, it has been suggested that after 

cleavage from NAPE, anandamide is immediately released from the 

membrane with the assistance of a membrane transporter (such as a 

P-glycoprotein) (Ayotte, Picone, and Makriyannis; unpublished results) or 

a lipid binding protein (like a lipocalin) (Piomelli, 1998). Such a lipid 

binding protein may also facilitate the passage of anandamide through the 

aqueous extracellular space to its sites of action. 

Endocannabinoid Inactivation 

Anandamide is inactivated in two steps, first by transport inside the cell 

and subsequently by intracellular enzymatic hydrolysis. The transport of 

anandamide inside the cell is a carrier-mediated activity, having been 

shown to be a saturable, time- and temperature-dependent process that 

involves some protein with high affinity and specificity for anandamide 

(Beltramo, 1997). This transport process, unlike that of classical neurotransmitters, 

is Na + -independent and driven only by the concentration 

gradient of anandamide (Piomelli, 1998). Although the anandamide transporter 

protein has not been cloned yet, its well characterized activity is 

known to be inhibited by specific transporter inhibitors. Reuptake of 2-AG 

is probably mediated by the same facilitating mechanism (Di Marzo, 

1999a,b; Piomelli, 1999). 

Once inside the cell, anandamide is hydrolyzed by a specific hydrolase, 

anandamide amidase (AEAase) or fatty acid amidohydrolase 

(FAAH) (Desarnaud, 1995; Deutsch, 1993). This enzyme is membrane 

associated and shows significant specificity for anandamide (Desarnaud, 

1995; Lang, 1999). 

There is some evidence that in cells with low anandamide amidase 

activity, such as platelets and neutrophils, anandamide is inactivated by an 

oxidative pathway involving 12(S)-lipoxygenase (Edgemond, 1998). 

Metabolism of anandamide by enzymes of the arachidonic acid cascade 


(Fig. 6) may be of physiological importance and may lead to possible 

biologically active oxygenated anandamide analogs. These pathways have 

not been explored yet. Nevertheless, it was shown that the 11-, 12-, and 15lipoxygenases 

recognize anandamide and catalyze its hydroxylation in 

vitro (Hampson, 1995). Among the resulting oxygenated anandamides, 

only the product of 11-lipoxygenase showed affinity to CB1 comparable to 

that of anandamide (Hampson, 1995). The physiological relevance of this 

finding, if any, is unknown at present. 

Less explored is the role and metabolic fate of 2-AG. It is possible 

that in many tissues, 2-AG is only an intermediate of a signaling pathway 

that generates 1,2-diacylglycerol and arachidonic acid, two well-known 

signaling molecules. In the brain however, 2-AG may have regulatory 

roles, since it escapes immediate metabolism and accumulates in response 

to stimuli-generated Ca 2+ surges (Stella, 1997). These differences may arise 

Figure 6 Anandamide metabolism: NAPE, N-arachidonylphosphatidyl-ethanolamides; 

PLD, phospholipase D; AEA, anandamide; AC, anandamide carrier 

protein; AT, anandamide transporter; AEAase, anandamide amidase; AA, 

arachidonic acid. 


from differences in the involved isoenzymes or their levels of expression 

from tissue to tissue. 

Anandamide amidase recognizes and hydrolyzes 2-AG (Goparaju, 

1999; Di Marzo, 1999; Lang, 1999); however, there is evidence for the 

existence of another specific hydrolase [monoacylglycerol (MAG) lipase] 

that hydrolyzes 2-AG (D. Piomelli and A. Makriyannis, 2000, personal 

communication). In addition to this pathway, 2-AG diffuses rapidly into 

the cell membrane where it could be either hydrolyzed to arachidonic acid 

and glycerol or esterified back to phosphoglycerides (Di Marzo, 1999b). 

IV. THE ENDOCANNABINOID SYSTEM 

It is apparent that a series of critical research breakthroughs during the last 

decade have unveiled a new significant biological assemblage, the endocannabinoid 

system. This system, which is evolutionarily well conserved, 

consists of at least two receptor types, each with different localization and 

functions; a family of endogenous ligands; and a specific molecular 

machinery for the synthesis, transport and inactivation of these ligands. 

Although information about this system is now emerging, many significant 

questions still remain unanswered. The anandamide transporter and some 

of the endocannabinoid metabolic enzymes have yet to be cloned. The 

accomplishment of a highly quantitative and detailed mapping of the 

endocannabinoid system will produce more information about its physiological 

roles. The advent of specific cannabinoid receptor antagonists 

(Pertwee, 1995c), (Rinaldi-Carmona, 1994) has already facilitated pharmacological 

studies by enabling reversal of the endogenous cannabinoid 

tone, as well as verification of the interaction of various agents with these 

receptors. The first inhibitors of anandamide amidase (Boger, 2000; 

Deutch, 1997a; Deutch, 1997b; Pertwee, 1995b) and its transporter (Beltramo, 

1997; Christie, 2001; Wilson, 2001) are becoming important tools in 

understanding the functions of the endocannabinoid system by producing 

an hypercannabinoid state. Thorough understanding of this system and its 

functions in physiological and disease conditions will likely lead to the 

development of new therapeutics. 

Invaluable tools for such studies are selective agents capable of 

interacting with the protein members of the cannabinoid system and, in 

turn, either activating or inhibiting them. Therefore, the study of the SAR 

of each of these targets and the identification of differences in ligand 

recognition comprises a task of great significance, one that can lead to the 

development of highly selective cannabinergic agents. The term ‘‘canna- 


Figure 7 Cannabinoid targets for drug design. 

binergic’’ encompasses ligands that act on proteins of the endocannabinoid 

system, regardless of chemical classification or type of resultant 

pharmacological activity. Therefore, this general term includes agents that 

act on the cannabinoid receptors, either as agonists or antagonists, as well 

as molecules that inhibit AEAase or the anandamide transporter (AT). The 

therapeutic potential that emanates from modulating these proteins 

renders them important yet unexploited targets for drug design and 

development (Fig. 7). 

All the aforementioned protein members of the cannabinoid system 

are large, membrane-bound proteins; therefore, it is particularly difficult to 

obtain direct information about their tertiary structure. Thus, at the 

present time, structure-based drug design is not feasible. Detailed exploration 

of the SAR and subsequent ligand-based design are the most appropriate 

means for the development of molecular probes for these proteins. 

V. MAJOR CLASSES OF CANNABINERGIC LIGANDS 

Based on chemical structure, cannabinergic ligands are classified into five 

major classes. Structures of representative members from each of the five 

chemical classes are shown in Figures 8 to 11. 

A. Classical Cannabinoids 

Classical cannabinoids (CCs) are tricyclic terpenoid derivatives bearing 

a benzopyran moiety. This class includes the natural product (–)-deltanine-tetrahydrocannabinol 

(Fig. 8, 1) and the other pharmacologically 

active constituents of the plant Cannabis sativa. 


Figure 8 Structures of representative classical cannabinoids. 

Figure 9 Nonclassical cannabinoids (NCCs). 


Figure 10 Representative cannabinergic aminoalkylindoles. 

Many classical cannabinoid analogs have been synthesized and 

evaluated pharmacologically and biochemically (Razdan, 1986; Mechoulam, 

1999). The CC structural features that seem to be important for 

cannabimimetic activity (Makriyannis, 1990) are as follows: 

1. The phenolic hydroxyl group, can be substituted by an amino 

group but not by a thiol group. In contrast to the traditional CC 

Figure 11 Structures of representative endocannabinoid analogs. 


SAR, which considers the phenolic hydroxyl to be one of the 

necessary pharmacophoric groups, analogs lacking it or bearing 

it in its etherified form retain high receptor binding affinity, (e.g., 

analog 2a) especially for CB2 (Huffman, 1996). 

2. The benzopyran ring is not essential for activity. The pyran 

oxygen can be substituted by nitrogen or can be eliminated in 

open-ring mono- or bisphenolic compounds. The recently 

developed CB2-selective ligand HU-308 (5) is an example of 

such a bicyclic cannabinoid (Hanus et al., 1999). 

3. Neither the double bond nor the 9-methyl group are necessary 

for activity. 

4. The alkyl chain is probably the most essential CC pharmacophoric 

group. Increased biological activity results from 

elongating the five-carbon delta-eight-THC chain to a sevencarbon 

chain substituted with 1 V ,1 V - (e.g., 2) or1 V ,2 V -dimethyl or 

with 1 V ,1 V -cyclic moieties (e.g., 3, AMG3). Oxygen atoms 

(ethers) and unsaturation (Papahatjis, 1998) within the chain, 

or terminal halogens, carboxamido, and cyano groups are 

well tolerated (Khanolkar, 2000). 

5. An additional pharmacophore introduced in the nonclassical 

cannabinoid series is the southern aliphatic hydroxyl (Makriyannis, 

1990). A variation involves the highly potent classical/nonclassical 

cannabinoid hybrids (e.g., 4, AM919) (Drake, 1998). 

B. Nonclassical Cannabinoids 

A second class of cannabimimetics was developed at Pfizer, in an effort to 

simplify the structure of CCs while maintaining or improving activity 

(Johnson, 1986). This class includes bicyclic (e.g., 6) and tricyclic (e.g., 7) 

analogs lacking the pyran ring of CCs (Fig. 9). These compounds are 

collectively specified as ‘‘non-classical cannabinoids’’ (NCCs). The crystalline 

CP55,940 (6) and its tritiated analog show high affinity, efficacy, and 

stereoselectivity to both cannabinoid receptors and have been used extensively 

as pharmacological tools. The key compound that led to the 

discovery of CB1 was [ 3 H]CP55,940 (Devane, 1988). 

The structural resemblance of NCCs and CCs, as well as their 

comparable SARs, indicate that they bind to CB1 in a similar fashion. 

The side chain and the phenolic hydroxyl of an NCC are crucial for 

activity. The hydroxypropyl chain of CP55,940 is not necessary for 


activity. However, when present, its stereochemistry is important and 

shows a strong preference for the beta relative configuration. 

C. Aminoalkylindoles 

The third chemical class of cannabinergics is that of aminoalkylindoles 

(AAIs) (Fig. 10). They were developed at Sterling Winthrop as potential 

nonsteroidal anti-inflammatory agents (Bell, 1991). These first analogs 

exhibited antinociceptive properties that were eventually attributed to 

interactions with the cannabinoid receptors. Compound 8 (WIN55212) 

is a potent CB1 and CB2 agonist with high stereoselectivity and a slight 

preference for CB2. AM630 (9), the first CB2-selective antagonist derived 

from this class of compounds, was developed in our laboratory after longterm 

efforts to obtain such an inhibitor (Pertwee, 1995a). We have recently 

reported the development of AM1241, a potent, highly CB2-selective 

agonist (Malan, 2001). 

This class of compounds differs from the first two by being considerably 

less lipophilic and more ‘‘druglike.’’ Labeling of CB1 with electrophilic 

AAIs almost abolished the receptor’s ability to bind to CP55,940, 

indicating that AAIs and NCCs (as well as CCs) share at least some points 

of interactions with CB1 (Yamada, 1996). Several models have attempted 

to define the pharmacophoric equivalency between the functional groups 

of AAIs, NCCs, and CCs (Xie, 1995), (Huffman, 1994). Although these 

three different classes of cannabimimetics show similarities in their 

binding with CB1, they differ considerably in the susceptibility of their 

binding affinities to different Na + -modulated allosteric receptor states 

(Houston, 1998). They also differ in their affinities to several CB1 

mutants (Chin, 1998), as well as in the way they activate the receptor 

(Houston, 1998). These differences may be explained by the existence of 

more than one ligand binding motif, or by ligand binding to partially 

overlapping but distinct receptor binding subsites, or even by induction 

of different receptor conformational changes upon binding of different 

ligands (Howlett, 1998a). It has been proposed that structurally dissimilar 

ligands may evoke different receptor–G-protein coupling (Houston, 

1998). Therefore, analogs from different cannabinoid ligand classes may 

evolve as selective pharmacological agents exhibiting only specific cannabimimetic 

effects. 

Structural features of AAI important for cannabinergic activity are 

the 3-aroyl moiety and the 1-chain, which must contain nitrogen, most 

often in a heterocyclic ring (e.g., piperidino or morpholino). This chain can 


e conformationally restricted as part of a six-membered ring fused to the 

indole nucleus (D’Ambra, 1992). 

D. Endocannabinoids 

The class of the endogenous cannabinoids (endocannabinoids) was discovered 

in 1992 as molecules produced by mammalian cells with affinity for 

the cannabinoid receptor (Devane, 1992). This class includes lipid molecules 

such as fatty acid ethanolamides, monoacylglycerols, and related 

synthetic analogs. The two prototypes in this class are the ethanolamide of 

arachidonic acid (anandamide) and 2-arachidonyl glycerol (2-AG). Its (R)- 

1V-methylated analog, AM356 (10) (Fig. 11) shows higher affinity and 

remarkable metabolic stability (Abadji, 1994). This analog, named Rmethanandamide, 

has been established as a standard CB1-selective agonist 

in the cannabinoid field. The (R,R)-2,1V-dimethyl anandamide was 

reported recently to exhibit a threefold improved affinity over R-methanandamide 

and significant enantioselectivity (Goutopoulos, 2001). Other 

modifications that result in high CB1 affinity include the substitution of the 

hydroxyl group with halogen, or the methyl group, and the substitution of 

the terminal n-pentyl chain with the dimethylheptyl chain, reminiscent of 

potent classical cannabinoid ligands (e.g., 12, O-1064) (Pertwee, 2000). 

This compound class also includes some fatty acid analogs designed for 

endocannabinoid targets other than the cannabinoid receptors. For 

instance, arachidonyltrifluoromethylketone (ATFMK) (13) and hexadecylsulfonyl 

fluoride (14, AM374) are potent inhibitors of anandamide 

amidase. The first inhibitor of the anandamide transporter to play an 

important role in the discovery of this transport process was AM404 (15) 

(Beltramo, 1997). 

E. 1,5-Biarylpyrazoles 

The fifth class, 1,5 biarylpyrazoles, was developed at Sanofi in 1994 from a 

hit generated by high throughput screening for cannabinoid receptor 

ligands (Rinaldi-Carmona, 1994). Compounds of this class act as cannabinoid 

receptor antagonists. Figure 12 shows SR141716A (16), which was 

reported, simultaneously with AM630, as the first CB1 antagonist and has 

since been used extensively as an important pharmacological tool. 

SR141716A shows selectivity for CB1 and often acts as an inverse agonist 

rather than a pure antagonist (Pertwee, 2000). Also developed at Sanofi, 

SR144528 (17) acts as an antagonist/inverse agonist with selectivity for 

CB2. A useful radioimaging agent in PET and SPECT studies [ 123 I] 


Figure 12 1,5 Biarylpyrazole cannabinoid receptor antagonists. 

AM281, a 123 I-labeled 1,5-biarylpyrazole was synthesized in our laboratory 

(Gatley, 1998). 

VI. THERAPEUTIC POTENTIAL OF CANNABINERGIC 

AGENTS 

Most known cannabimimetics today have very broad effects on organ 

systems, several of which are still not completely delineated. The ubiquitous 

pharmacology of cannabimimetics is one of the reasons for the 

failure, thus far, of the clinical application of these drugs to reach its full 

potential. The sections that follow summarize the effects of cannabinergics 

on the various physiological systems and the possible therapeutic uses that 

may arise from these biological activities. 

A. Nervous System 

The primary system of cannabimimetic activity is the nervous system. The 

CB1 receptor is omnipresent in the brain, especially in areas that control 

functions affected by cannabimimetics. One of the functions most pronouncedly 

influenced by cannabimimetics is motor behavior. Catalepsy, 

immobility, ataxia, and impairment of complex behavioral acts after acute 

administration of high doses of cannabimimetics are manifestations of 

such motor effects (Pertwee, 1997). In lower doses cannabimimetics 

produce the opposite effects. The very dense presence of CB1 in the 

cerebellum and the basal ganglia, areas responsible for motor activity, is 


congruent with these observations. The GABA function in the basal 

ganglia is enhanced by CB1 agonists (Consroe, 1998). Cannabimimetics 

seem to exert an important modulatory action in basal ganglia output 

nuclei by inhibiting both inhibitory striatal input, which is tonically 

inactive, and excitatory subthalamic input, which is tonically active 

(Sanudo-Pena, 1999). The net cannabimimetic effect on motor activity 

depends on the level of activity of each of these two functions. This may 

explain the biphasic effect of cannabimimetics on motor behavior. 

An important recent discovery has advanced the current understanding 

of how cannabimimetics are implicated in the control of motor 

behavior (Giuffrida, 1999). Giuffrida et al. have reported that D2 activation 

in the striatum results in release of the endocannabinoid anandamide, 

which in turn seems to mediate a negative feedback control, counteracting 

dopamine-induced facilitation of motor activity (Giuffrida, 1999). Because 

of these effects of cannabinergics on the basal ganglia and subsequently on 

motor activity, it has been suggested that cannabinergics may be useful 

agents in the treatment of motor disorders such as choreas, Tourette’s 

syndrome, dystonias, and Parkinson’s disease (Consroe, 1998). In general, 

by increasing hypokinetic features in the basal ganglia, CB1 agonists may 

alleviate the various hyperkinetic manifestations, such as choreic movements, 

that characterize basal ganglia disorders. Direct evidence suggesting 

the involvement of CB1 in Huntington’s chorea is the extensive loss of 

CB1 receptors in the substantia nigra and lateral globus pallidus (Glass, 

1993). It is still unclear whether these observations are causative of 

Huntington’s disease or its results. However, this finding alone argues that 

a suitable CB1 ligand could potentially be useful as a diagnostic agent for 

this chorea. 

Furthermore, the presence of CB1 in the structures and pathways 

associated with the pathophysiology of Tourette’s syndrome, and especially 

the functional link between CB1 and D1, D2, also argues that the 

endocannabinoid system may have some involvement in this disorder as 

well (Consroe, 1998). In addition, it has been suggested that activation of 

CB1 receptors, also owing to their link with the dopaminergic system, may 

reduce dyskinesia produced by L-DOPA in patients with Parkinson’s 

disease (Brotsie, 1998). 

The CB1 receptors present in the hippocampus, amygdala, and 

cerebral cortex may be responsible for observations that cannabimimetics 

are effective against some types of seizures (Consroe, 1998). The anticonvulsant 

and antispastic effects of cannabinoids are well documented, 

however the mechanisms of these effects are still unclear (Nahas, 1999). 


Orally administered cannabimimetics can relieve some of the symptoms of 

multiple sclerosis and spinal cord injury such as muscle spasticity, pain, 

tremor, nystagmus, and nocturia (Pertwee, 2000). Recent studies (Baker, 

2000; Baker, 2001) have shown that exogenously administered cannabimimetics 

control spasticity in a multiple sclerosis (MS) model. Possible 

implication of both CB1 and CB2 receptors has been suggested. Agents 

that elevate anandamide levels by inhibiting AEAase (AM374) or AT 

(AM404) also produced these antispastic effects indirectly. Cannabinoid 

receptor antagonists blocked these antispastic effects. Respectively, 

SR141716A and SR1445228, selective CB1 and CB2 antagonists/inverse 

agonists, produced enhanced spasticity when administered alone to the 

same animal model (Baker, 2000). Furthermore, it was evident that 

endocannabinoids are released during episodes of MS, during which they 

alleviate the spastic effects of the disease (Pertwee, 2000). These findings 

confirm, at least to some extent, the anecdotal reports that marijuana 

smoking alleviates the symptoms in MS patients and establishes cannabimimetics 

as exciting candidates for the development of agents that control 

spasticity and other abnormalities resulting from some neurodegenerative 

diseases. These agents may also control spasticity produced by spinal cord 

injury by acting on spinal as well as on supraspinal mechanisms (Consroe, 

1998). It has been suggested that the effect of cannabimimetics on the 

release of glutamate in the substantia nigra appears to be the most 

important supraspinal mechanism of cannabimimetic-induced control of 

spasticity (Consroe, 1998). 

The CB1-mediated inhibition of glutamate release in the hippocampus 

was also suggested to be the most likely mechanism of the neuroprotective 

effects of WIN5521,2 observed in both the global and focal 

cerebral ischemia animal models (Nagayama, 1999). These effects were 

stereoselective and were blocked by SR141716A. Therefore, cannabimimetics 

may find potential therapeutic utility in the treatment of disorders 

resulting from cerebral ischemia, including stroke. 

Another neuroprotective activity of cannabimimetics was shown to 

be associated with the CB1-mediated inhibition of nitric oxide (NO) release 

from rat microglial cells (Waksman, 1999). This study suggests cannabimimetics 

as potentially useful agents in brain injury resulting from 

inflammatory neurodegenerative processes, especially those involving 

activation of microglial cells, such as AIDS-encephalitis. 

Another significant cannabinoid activity that is mediated by the 

nervous system arises from the antinociceptive properties of these agents. 

Compelling evidence suggests that cannabimimetics are effective in the 


control of acute and chronic pain in a variety of antinociceptive tests in 

animals (Martin, 1998). Synthetic cannabimimetics have been classified as 

equal to morphine in potency and efficacy (Walker, 1999). The mechanism 

of the cannabimimetic-induced analgesia is multifaceted and occurs at 

several levels: (1) directly on spinal cord mechanisms (Walker, 1999); (2) in 

supraspinal mechanisms, specifically in the thalamus and the periaqueductal 

gray (PAG) matters (Walker, 1999; Martin, 1998); and (3) in the 

periphery, possibly involving CB1-like and CB2-like receptors (Calignano, 

1998). Other systems, such as n and A opiate receptors, as well as spinal 

noradrenergic mechanisms, seem to be involved in the cannabimimeticproduced 

analgesia (Walker, 1999). Evidence supports the suggestion that 

cannabimimetics are effective in animal models of chronic pain, a type of 

pain that is poorly managed by opioids (Walker, 1999). It has also been 

suggested that CB1 agonists may be superior to morphine in suppressing 

pain caused by nerve damage (Pertwee, 2000). This type of pain is signaled 

by abnormal discharges of Ah and Adelta fibers, which are much more 

populated by CB1 than A-opioid receptors. 

Another category of CNS-mediated cannabinoid effects includes 

alterations in cognition and memory. Cannabimimetics have been shown 

to interfere with the mechanisms of long-term potentiation (LTP), a 

candidate mechanism for learning and memory. They also alter presynaptic 

release of GABA and glutamate from hippocampal neurons (Hampson, 

1998). Hippocampus, a structure rich in CB1, plays a major role in memory 

processing, especially by enabling memory retrieval, whereas retrohippocampal 

areas with fewer CB1 receptors are responsible for memory 

storage. Hippocampal lesions in rodents impair short-term memory. 

Several behavioral studies have exhibited that cannabinoids disrupt information 

processing in the hippocampus, acting as ‘‘reversible’’ hippocampal 

lesions (Hampson, 1999). It is suggested that the role of CB1 in 

these regions is to regulate storage information by switching hippocampal 

memory circuits (Hampson, 1998). The role of the cannabinoid 

system in memory and cognition renders it a possible target for memory 

and cognition enhancing agents. This possibility is strongly supported 

by some recent advances in understanding the neurobiology of the 

endocannabinoid system (Wilson, 2001; Christie, 2001; Kreitzer, 2001; 

Ohno-Shosaku, 2001). Endocannabinoids were found to be the neurotransmitters 

responsible for the depolarization-induced suppression of 

inhibition (DCI) and excitation (DCE). Since DCI enhances memory in 

the hippocampus, drugs that inhibit the metabolism and especially the 

transport of endocannabinoids are very likely to have a beneficial effect on 


memory by increasing the levels of endocannabinoids at the sites where 

DCI takes place (Christie, 2001). Direct cannabinoid receptor agonists 

flood the endocannabinoid system, resulting in the well-known overall 

disruptive effect in memory and cognition. 

Cannabinoids are long known for their psychoactive and euphoric 

‘‘high’’ effects and have been used for these properties for centuries. Their 

addictive potential and mechanisms appear to be qualitatively and quantitatively 

different from those of other drugs of abuse. However, recent 

studies indicate that cannabimimetics, similar to other addictive drugs, 

activate the brain reward/reinforcement circuit (ventral tegmental area, 

nucleus pallidus, and ventral pallidum) and produce reward-related 

behaviors in laboratory animals (Gardner, 1998). Efforts to separate these 

unwanted effects from the desired ones have had only limited success thus 

far. This fact, along with the negative social perception of these drugs, has 

been a major hindrance to the development of cannabinergic therapeutics. 

However, the increasing understanding of the endocannabinoid system 

presents us with possibilities for the design of selective agents. Indirect 

activation of this system by increasing endocannabinoid levels only at the 

sites where they are physiologically produced through inhibition of 

endocannabinoid catabolism or transport may lead to increased selectivity 

and fewer undesired effects than activation of the cannabinoid receptors 

with direct agonists (Pertwee, 2000). Endocannabinoids such as anandamide 

were shown to have a much lower physical dependence potential 

(Aceto, 1998). 

Other well-known central cannabimimetic effects that nevertheless 

are not well understood are hypothermia, appetite stimulation, and 

antiemetic effects. Cannabimimetic-induced hypothermia is thought to 

occur by decreasing the thermoregulatory set point through interactions 

with the relevant hypothalamic centers (Pertwee, 1995b). Cannabimimetics 

also stimulate hunger in humans and animals, particularly for solid, 

sweet tasting foods (Pertwee, 1995b). For this property, delta-9-THC 

(marinol) is clinically used today for the management of AIDS-wasting 

syndrome (Nahas, 1999). The advent of potent and CB1-selective ligands 

lacking the CB2-mediated immunosuppressive properties may present 

significant advantages over the currently used delta-9-THC in the treatment 

of AIDS patients who are already severely immunocompromised. It 

is also conceivable that cannabinoid receptor antagonists may be proven 

effective as appetite suppressants, as suggested by the results of a study 

showing that SR141716A, a selective CB1 antagonist/inverse agonist, 

suppressed rodent appetite for sucrose and ethanol (Arnone, 1998). 


A second current clinical indication of cannabimimetics is their 

antiemetic and antinausea effects, especially in cancer chemotherapy 

patients. These effects are mediated above the level of vomiting reflex 

and possibly through descending inhibitory connections to the lower brain 

stem centers (Levitt, 1986). 

B. Immune System 

The discovery of the peripheral CB2 receptor, which localizes in cells of the 

immune system, is very likely linked to the well-known immunosuppression 

of marijuana smokers. 

Miskin (1985) found that delta 9-THC decreases host resistance to 

herpes virus type 2 in mice and guinea pigs by decreasing both cellular and 

humoral immunity. In vivo and in vitro studies indicate that macrophages 

are the major targets of cannabinoids. delta 9-THC inhibits, in a dosedependent 

manner, the extrinsic antiviral activity of macrophages (Cabral, 

1991). It was also shown that cannabinoids cause morphological changes 

in macrophages (Cabral, 1991) and affect their phagocytic and spreading 

ability (Spector, 1991). 

The involvement of CB2 (and possibly of CB1) receptor(s) in the 

immunosuppressive effects of cannabinoids is not proven yet. The localization 

of CB2 in cells of the immune system and especially in macrophages 

and lymphocytes suggests that this receptor serves some immunoregulatory 

role(s). The first strong piece of evidence that implicates CB2 in such a 

function came from Kaminski et al. (1994), who demonstrated that 

cannabinoid-induced suppression of humoral immunity was partially 

mediated through inhibition of adenylyl cyclase by a G-protein-coupled 

mechanism that is pertussis toxin sensitive. Involvement of a membrane 

perturbation mechanism in cannabinoid-induced immunosuppression is 

also possible, especially in areas exposed to high drug concentrations, such 

as lung alveolar macrophages of marijuana smokers (Cabral, 1999). The 

involvement of the cannabinoid system in the regulation of the immune 

system may suggest that cannabinergics could potentially serve as immunomodulatory 

agents. Although CB2 selective agents already exist, their 

clinical potential in some immunomodulatory role will not be realized until 

the CB2 physiological functions are better understood. Cannabidiol, a 

cannabis terpenoid ingredient lacking the pyran ring as well as significant 

binding affinity for CB1 and CB2, was shown to be an active antiinflammatory 

agent in the murine model of arthritis (Pertwee, 2000). The 

molecular basis of this observation is still unknown. 


C. Cardiovascular System 

Cannabinoids reduce platelet aggregation and also produce tachycardia 

and orthostatic hypotension due to peripheral vasodilation. A distinct, 

CB1-like, receptor is found in the endothelium of rat mesenteric arteries 

(Jarai, 1999). This receptor mediates a remarkable vasodilating effect after 

activation by any of several CCs, anandamide, or some CB1-inactive CClike 

analog. This effect is NO independent and is inhibited by the CB1 

antagonists, SR141716A and AM251 (Batkai, 2001), and also by cannabidiol. 

It is possible that exploitation of this new cannabinoid target may 

lead to new types of hypotensive agents. 

D. Reproductive System 

Cannabinoids produce increased ring and chain chromosomal translocations 

and morphological abnormalities in mouse sperm, as well as reduction 

of sperm concentration in humans (Zimmerman, 1999). Strong 

evidence indicates the presence of functional CB1, or CB1-like receptors, 

in human sperm (Schuel, 1999). Furthermore, the endogenous cannabimimetic 

anandamide is produced in the human uterus and testes (Schuel, 

1999). These findings along with several observations on cannabinoidinduced 

effects on reproductive functions suggest that the cannabinoid 

system may be directly involved in the regulation of sperm production, 

sperm motility, the acrosome reaction, and prevention of polyspermy 

(Schuel, 1999). The endocannabinoid system in the uterus appears to play 

a fundamental role in embryo implantation and early development. 

Anandamide inhibits these processes and, therefore, regulation of its 

levels seems to control the timing of these events (Paria, 1995). These 

findings are also in line with recent clinical observations that correlate the 

levels of AEAase expression with miscarriages in pregnant women 

(Maccarone, 2000). Further understanding of the endocannabinoid functions 

in the reproductive system will open perspectives for exploitation of 

cannabinergics for the treatment of some types of infertility or the development 

of contraceptives. 

Cannabimimetics are also shown to affect reproductive and 

metabolic functions indirectly by hormonal modulation through the 

hypothalamic and pituitary regulatory centers. They are found to 

reduce serum levels of the luteinizing hormone, prolactin, growth 

hormone, and thyroid-stimulating hormone, and to increase corticotropin 

(Murphy, 1998). 


E. Eye 

Cannabinoids reduce intraocular pressure, probably by directly affecting 

ocular fluid outflow pathways. The mechanism of this effect is unknown, 

and its link to cannabinoid receptors has yet to be established (Green, 

1999). Marijuana smoking is allegedly helpful to glaucoma patients, and 

the potential use of cannabimimetics for the treatment of glaucoma has 

long been recognized. New formulation technologies, as well as the advent 

of less hydrophobic cannabimimetics, present us with opportunities to 

overcome the challenge of local drug delivery to the eye. 

F. Respiratory System 

Cannabimimetics are known to produce bronchodilation, which is manifested 

by a marked increase in airway conductance and reduction in 

airway resistance (Vachon, 1973). Although the mechanism of this activity 

is not known, it probably does not directly involve adrenergic receptors. 

Possible involvement of CB1A (a CB1 variant found in the lung) in 

cannabinoid-induced bronchodilation is still unexplored (Shire, 1995). 

Recently, it was shown that anandamide is released in the lung upon 

Ca 2+ stimulation and exerts a dual effect on bronchial response. It strongly 

inhibits capsaicin-evoked bronchospasm and cough; however, it causes 

bronchoconstriction in vagotomized rodents (Calignano, 2000). These 

effects are mediated by CB1 receptors present in axon terminals of airway 

nerves since they are blocked by SR141716A. This endocannabinoidmediated 

control of airway responsiveness may be exploited in the development 

of new antiasthmatic agents. 

G. Gastrointestinal System 

Cannabimimetics reduce the intestinal motility by a CB1-mediated inhibitory 

activity on acetylcholine release from autonomic fibers. An endocannabinoid, 

2-AG, was isolated from dog intestine; however, its role there 

remains unknown (Mechoulam, 1995a). 

VII. CONCLUSIONS 

With the discovery of anandamide and 2-arachidonyl glycerol as two new 

families of endocannabinoids, cannabinoid research has taken major 


strides toward arriving at an understanding of the molecular mechanism of 

cannabinoid action. Currently, there are multiple characterized endocannabinoid 

proteins [at least two receptors, CB1 and CB2; an enzyme, 

arachidonylethanolamide amidohydrolase (AEAase); and a transport 

protein, anandamide transporter (AT)] as potential therapeutic targets 

for the development of useful medications in the treatment of a multitude 

of conditions such as drug addiction, pain, and motor disorders. A number 

of ligands (receptor-selective agonists/antagonists, inverse agonists, 

enzyme inhibitors, transport inhibitors) are also available which can serve 

as important research tools for exploring the endocannabinoid biochemical 

pathways and their role in the modulation of behavior, memory, 

cognition, and pain perception. This is significant progress, considering 

that only about a decade ago the sites of action of cannabinoids had not yet 

been identified and their molecular mechanism of action was still under 

question. The future of endocannabinoid research is undoubtedly very 

exciting and full of promise. 

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5 

Development of PET 

and SPECT Radioligands 

for Cannabinoid Receptors 

S. John Gatley, Andrew N. Gifford, and Yu-Shin Ding 

Brookhaven National Laboratory, Upton, New York, U.S.A. 

Nora D. Volkow 

NIDA, Bethesda, Maryland, U.S.A. 

Ruoxi Lan, Qian Liu, and Alexandros Makriyannis 

University of Connecticut, Storrs, Connecticut, U.S.A. 


Marijuana is the most commonly illegal drug of abuse in the United 

States, but relatively little known about how activation of cannabinoid 

receptors leads to the psychoactive effects desired by its abusers, or 

whether receptor densities are altered in addiction or detoxification, or in 

other disease states. Futhermore, the two known G-protein-coupled 

receptors for cannabinoids (CB1, which is found in brain and some 

peripheral tissues, and CB2, whose distribution is believed to be restricted 

to immunological tissues in the periphery) are potentially important 

targets for drug development. Various dosage forms of D 9 -tetrahydrocannabinol 

(THC), the major psychoactive constituent of marijuana, as well 

as novel compounds, are under investigations by several major drug companies. 

For example, recently developed subtype-selective antagonists 

of cannabinoid receptors such as SR141716A may have beneficial drug 


actions in treating memory impairment and other disorders. A publication 

by the Institute of Medicine/National Academy of Sciences entitled 

‘‘Marijuana and Medicine: Assessing the Science Base’’ discusses some of 

the issues involved in this area. 

Positron emission tomography (PET) is an imaging method used to 

measure the regional distribution and kinetics of chemical compounds 

labeled with short-lived positron-emitting isotopes such as 11 C (half-life = 

20 min) and 18 F (110 min). It thus enables direct measurement of 

components of the neurochemical systems in the living human brain 

[1,2]. The SPECT (single-photon emission-computed tomography) methodology 

can also be used to measure some of the same components of 

neurochemical systems as PET. As discussed later, SPECT is inferior to 

PET in terms of spatial resolution, sensitivity, and quantitation. On the 

other hand, SPECT methodology is less expensive than PET and is far 

more widely available because of its advantages in clinical nuclear 

medicine. These include the use of radionuclides of longer half-life, such 

as 123 I (13 h) and 99m Tc (6 h). 

Both PET and SPECT have been used in studies of several drugs to 

image functional consequences of acute or chronic drug treatment, using 

radiotracers that measure changes in factors such as blood flow, glucose 

metabolic rate, or dopamine release. They have also been used to image 

changes in the apparent brain concentrations of neuroreceptors with which 

the drugs of abuse directly or indirectly interact. For a review of this area, 

see Gatley and Volkow [3]. The brain–dopamine system, which may be 

involved in the actions of all drugs of abuse, has been an important target 

of PET studies [4]. 

II. IMAGING THE EFFECTS OF CANNABINOIDS 

ON METABOLISM AND BLOOD FLOW 

Relatively few human imaging studies have evaluated the effects of marijuana 

or THC on metabolism or blood flow. Acute intravenous THC in 

both normal controls and habitual marijuana users led to increased an 

increased regional cerebral metabolic rate (CMR) in the cerebellum. This 

increase is positively correlated both with concentrations of THC in the 

plasma and with the intensity of the subjective sense of intoxication [5]. 

In a 1997 PET/[ 15 O]water study with 32 abusers [6], THC dose-dependently 

increased cerebral blood flow (CBF) in the frontal regions, insula 


and cingulate gyrus, and subcortical structures, with somewhat greater 

effects in the right hemisphere. Self-ratings of THC intoxication were 

correlated most markedly with the right frontal region. Most subjects 

exhibited increased rCBF in cerebellum. However, those whose cerebellar 

CBF decreased also had a significant alteration in time sense [7]. 

The average increase in rCMR after THC administration was less in 

marijuana users than in controls, and users had lower cerebellar metabolism 

than the controls at baseline [8]. Thus the cerebellum shows the 

greatest metabolic increase in response to acute THC and responds to 

chronic marijuana exposure with a decrease in baseline CMR. Habitual 

users but not controls responded to THC administration with increased 

rCMR in prefrontal cortex, orbitofrontal cortex, and basal ganglia. In contrast 

to the robust effects of THC on relative rCMR, changes in global 

CMR in response to THC were quite variable, with increases, decreases, 

and no changes seen in equal numbers of subjects. There was also variability 

in subjective effects, which were pleasurable for most subjects but 

either minimal or unpleasant (anxiety or paranoia) for others. 

The involvement of the cerebellum in the psychoactive effects of 

marijuana and in changes in rCMR is consistent with the view that THC 

interacts with the high concentration of CB1 receptors in this brain area. 

Decreases in the cerebellar rCMR in habitual marijuana users may reflect 

the effects of chronic exposure to the drug. Functions known to be 

associated with the cerebellum, such as motor coordination, proprioception, 

and learning, are adversely affected both during acute marijuana 

intoxication and in habitual users. 

The cannabinoid CB1 receptor is the binding site in the brain for 

D 9 -tetrahydrocannabinol, the active principle of marijuana. This Gprotein-coupled 

receptor is abundant in specific brain areas including 

the cerebellum, the hippocampus, and the outflow nuclei of the basal 

ganglia. An in vivo radioligand for the CB1 receptor would allow us to 

evaluate disease and drug-induced changes in cannabinoid receptor 

densities, and possibly to investigate relationships between receptor 

occupancy by agonist and antagonist ligands and their behavioral and 

toxic effects. Such studies would contribute not only to our understanding 

of the neural basis of marijuana abuse, but also to medication 

development, since pharmacological manipulation of the CB1 receptor 

system might prove useful in conditions such as chronic pain and 

multiple sclerosis. In addition to compounds with the classical cannabinoid 

skeleton, recently developed high affinity agonists and antagonist of 

the brain cannabinoid receptor may serve as lead compounds for PET 


Figure 1 Structures of representatives of classes of compounds that blind to 

cannabinoid receptors. 

and SPECT radiotracers. These have nonclassical cannabinoid, aminoalkylindole, 

anandamide, and pyrazole strructures (Fig. 1) [9–13]. 

Autoradiographic studies with tritiated CP55,940 and other high 

affinity agonists (see, e.g., Ref. 14) demonstrated high concentration of 

cannabinoid receptors in the basal ganglia and especially in its outflow 

nuclei, the globus pallidus and the substantia nigra. High concentrations 

are also found in the hippocampus and the cerebellum. The cerebral 

cortex also contains appreciable concentrations of cannabinoid receptors, 

the highest being the cingulate gyrus. Some other regions including 

most of the brain stem and the thalamus, contain low or negligible 

concentrations. The pattern of distribution of cannabinoid receptors in 

many brain regions is similar to that of dopamine D1 receptors, which 

has led to the suggestion that a function of the cannabinoid system may 

be to induced modulate brain dopaminergic activity [15]. 

III. ATTEMPTS TO DEVELOP RADIOLIGANDS 

The first attempt to develop a PET radioligand for imaging brain CB1 

receptors involved modification of D 8 -THC by labeling with fluorine-18 in 

the hydrocarbon side chain [16,17], as shown in Figure 2. Unfortunately, 


this compound did not produce PET images that showed any particular 

regional pattern of brain localization when injected into a baboon. It 

showed poor uptake but was widely distributed in the brain. Clearance of 

radioactivity that did appear to enter the brain was rapid. Furthermore, 

uptake of radioactivity in the skull was apparent, which suggested in vivo 

decomposition of the radiotracer, leading to the production of labeled 

fluoride ion, which then accumulated in bone. It is likely therefore that the 

PET images represented only nonspecific uptake of the tracer with a 

negligible component due to specific binding to cannabinoid receptors. 

Studies with [ 18 F] D 8 -THC supported the view that a successful 

radiotracer must have adequate metabolic stability and a fairly high 

affinity for the CB1 receptor to ensure that radioactivity is retained in 

brain tissues long enough for tomographic measurement. Furthermore, 

a good radiotracer should exhibit high uptake into the brain. This is 

likely to be a difficulty for cannabinoid receptor radioligands, since 

these molecules are extremely lipophilic. High log P values are generally 

associated with poor blood–brain barrier penetrability, presumably 

because they remain dissolved in lipid structures in the blood during 

transit through the brain capillary bed. The nonclassical cannabinoid 

CP55,940, the aminoalkylindole WIN55, 212-2, and THC are reported 

to possess log P values of about 6, 5, and 7, respectively. Even the 

lowest log P value in this series (5) has been associated with poor brain 

penetration in other classes of molecules (see, e.g., Refs. 18, 19). 

Furthermore, although [ 3 H]WIN55,212-2 has an affinity about 10-fold 

higher than THC, it does not exhibit preferential localization in CB1receptor-rich 

areas of the mouse brain when injected intravenously 

(Gifford et al., unpublished). 

Figure 2 Incorporation of fluorine-18 into 5V-[ 18 F]fluoro-D 8 -THC. 


Figure 3 Relationships between structures of SR141716A, AM251, and AM281. 

The introduction of the diarylpyrazole CB1 receptor antagonist 

SR141716A [12] immediately suggested that exploitation of this class of 

molecules might lead to development of a successful CB1 receptor radioligand. 

Not only is the affinity of SR141716A of the order of 100-fold 

higher than that of THC, but the structure suggested that the log P value 

would be considerably lower. In addition, SR141716A is highly selective 

for the brain cannabinoid receptor (CB1) relative to the CB2 receptor 

found in cells of the immune system. It is also an antagonist, which is a 

potential advantage because, in the binding of antagonists of G-proteincoupled 

receptors, there is no discrimination between receptors in different 

affinity states. This is unlike the situation with agonists, which bind predominantly 

to a high-affinity state of the receptor. Finally, SR141716A 

contains three chlorine atoms, suggesting that replacement of one of these 

with a radioactive iodine atom might produce a compound with the desired 

properties. 

Our ‘‘mark I’’ pyrazole radioligand, code-named AM251, was synthesized 

in nonradioactive and radioactive forms [20,21]. Following 

intravenous injection in mice and rats, the radioiodinated compound 

concentrated preferentially in brain areas known to contain densities 

of CB1 receptors [22]; however, it failed to enter the brain in SPECT 

experiments conducted with baboons [23]. On the hypothesis that this 

failure was associated with too high a log P value, we synthesized a 

related, ‘‘mark II’’ radioligand with an additional structural modification. 

This was replacement of the piperidine ring of SR141716A and 

AM251 with the more polar morpholino ring (Fig. 3). This compound 

AM281, was able to visualize CB1 receptors in baboon (Fig. 4) and 

rodent (Fig. 5) brains in vivo [23]. 


Figure 4 Sagittal sections of the brains of two baboons injected intravenously 

with [ 123 I]AM251 (left) or [ 123 I]AM281 (right). These experiments indicated that 

AM281 is to penetrate the baboon brain much more readily than AM251. 

Figure 5 Ex vivo autoradiography of [ 123 I]AM281 in rat brain gave distribution 

patterns that were essentially identical to in vitro autoradiographs obtained using 

tritiated high affinity agonists. 


Figure 6 Comparison of the behavior of radioidinated AM251 and AM281 

in mice. 

The degree of failure of AM251 to enter the baboon brain, as assessed 

by SPECT scanning, was surprising because in rats and mice AM251 does 

clearly enter the brain. Figure 6 presents a comparison of AM251 and 

AM281 in mice. In absolute terms, the graphed data should be interpreted 

cautiously because the experiments with the two radioligands were not 

conducted simultaneously, and other experimental details were not identical 

[22,23]. However, apparently greater brain uptake of AM281 at early 

times is consistent with its smaller log P value. Moreover, it is clear that 

there was significant clearance of AM281 between 30 and 120 min, whereas 

there was no significant difference between the 30, 60, and 120 min data 

points for AM251. The in vivo brain uptake data, which show a more 

prolonged retention of AM251, were thus consistent with the in vitro 

binding data, which indicated that AM251 has an approximately threefold 

higher affinity for the CB1 receptor than AM281. The mouse data shown 

in Figure 6, however, did not predict the large difference seen in baboons 

(Fig. 4). It may be that tight binding of AM251 to a specific blood protein, 

rather than a greater distribution of AM251 into lipophilic blood components, 

is responsible for its low brain penetration in baboons. 


Although our SPECT studies with [ 123 I]AM281 provided a proof of 

principle that in vivo imaging of CB1 receptors in primates is possible, the 

behavior of this radioligand is far from ideal in that, relative to radiotracers 

used to study other neurochemical systems, its uptake in the brain is low 

and its clearance is rapid. These factors limit the count rate obtained in 

radionuclide imaging studies, and thus the quality of the images. Because 

PET is more sensitive than SPECT by an order of magnitude, a CB1 

receptor radioligand labeled with fluorine-18 with pharmacokinetic properties 

to those of similar AM281 would probably be quite acceptable for 

human use. On the other hand, a radioiodinated compound for use with 

SPECT would be more useful if it had at least the initial brain uptake of 

AM281, but a higher affinity, to ensure a longer clearance time. Since our 

initial published work with AM281, several other candidate radioligands 

have been prepared [24–26]. However, to our knowledge no reports of 

human studies have appeared. 

IV. ONGOING WORK 

In our own laboratories, we have continued to synthesize and evaluate new 

labeled cannabinoid receptor radioligands. One of these is [ 18 F]AM284, 

where the labeled atom is part of a fluoropentyl group on position 1 of the 

pyrazole ring (Fig. 7; see also Table 1). Although this (unpublished) study 

demonstrated in vivo binding of AM284 to CB1 receptors, as shown by 

the fact that co-injection with SR141716A reduced brain binding, the 

Figure 7 Structure of an 18 F-labeled pyrazole ligand; for brain uptake (see Table 

1). 


Table 1 Brain Uptake Data for the 18 F-labeled Pyrazole Ligand of Figure 7 

Injected activity in whole brain (%) a 

Conditions AM281 AM284 

15 min 0.80 F 0.05 0.07 F 0.008 

60 min (vehicle) 0.66 F 0.05 * 

0.028 F 0.004 ** 

60 min (+ SR14176A) 0.33 F 0.05 *** 

0.020 F 0.001 **** 

* p < 0.003; cf. 15 min time point. 

** p < 0.001; cf. 15 min time point. 

*** p < 0.001; cf. vehicle. 

**** p < 0.035; cf. vehicle. 

a Values are the mean FSD (n = 5). 

binding was less than one-tenth that of AM281 measured simultaneously 

in a dual-isotope experiment. It would therefore not be a practicable PET 

radioligand. Other fluorine-18 and radioiodinated cannabinoid receptor 

ligands are being synthesized and studied in our laboratories. 

While we have not yet started human PET or SPECT studies, we have 

used AM281 to conduct fundamental studies of the CB1 receptor system in 

Figure 8 Comparison of ability of WIN 55, 212-2 to sedate mice (triangles) and 

to block specific binding of [ 131 I]AM281 (squares). 


odents. For example, during experiments in mice designed to measure the 

occupancy of CB1 receptors associated with physiological effects of 

exogenous cannabinoids, we found that doses of WIN 55,212-2 that 

induced profound sedation did not reduce binding of AM281 to cerebellum 

and hippocampus (Fig. 8). This observation indicates that the 

occupancy of the CB1 receptor necessary for physiological effects of 

cannabinoids is very low [27]. 

Experiments in superfused hippocampal slices prepared from rats 

were then conducted to compare inhibition of acetylcholine release by the 

cannabinoid receptor agonist WIN55,212-2 with inhibition of AM281 

binding by this agonist. The results (Fig. 9) show that half-maximal 

response is achieved at less than 1% occupancy, confirming that the 

agonist occupancy necessary to produce a physiological response is very 

low in the CB1 system [27]. 

A consequence of a very large receptor reserve for CB1 receptors 

would be that PET or SPECT could not be used to image the occupancy of 

the CB1 receptor by biologically significant doses of agonist drugs. This is 

Figure 9 Graph of inhibition of acetylcholine release in superfused hippocampal 

slices versus CB1 receptor occupancy estimated from inhibition of 

[ 131 I]AM281 binding. 


ecause even high doses of an agonist would not displace an appreciable 

fraction of the radiotracer binding. On the other hand, these experiments 

indicated that the binding of exogenous CB1 receptor antagonist radioligands 

should not be affected by changes in the levels of endogenous 

ligands such as anandamide. This would thus remove a possible confounding 

factor in imaging experiments designed to detect changes in 

cannabinoid receptor densities. 

In the light of these experiments with WIN55,212-2, it is of interest to 

speculate on the degree of occupancy of brain CB1 receptors that is 

achieved by doses of THC that induce desired effects in human during 

the smoking of a marijuana cigarette. THC is a partial agonist with an 

efficacy of 20 to 25% [28,29], so that it would act at a higher receptor 

occupany than a full agonist like WIN55,212-2. Intravenous doses of 0.5 

mg/kg THC are effective in humans [30], and if 1% of the injected dose is 

distributed in the brain [31], this would correspond to a concentration of 

about 15 nmol/L. Applying the mass action equation and assuming that an 

in vitro Kd value for the CB1 receptor of 100 nM is appropriate, and a Bmax 

value of 100 nmol/L, an occupancy of 7% is estimated. However, it is likely 

that the fraction of THC available for binding to the receptor in vivo is 

quite small, since as an extremely lipophilic molecule, it will be distributed 

in brain membranes. This is expected to increase the effective Kd value in 

vivo and so lower the estimate of occupancy, possibly by more than one 

order of magnitude. These considerations, therefore, suggest that only a 

very small proportion of the brain CB1 receptors need be activated to 

induce psychoactive effects in humans, consistent with our results in mice, 

and that PET studies will not be able to measure the degree of occupancy 

achieved by marijuana smokers. 

Similar studies were done to evaluate the relationship between level 

of occupancy of the CB1 receptor by nonradioactive AM281 and the 

degree to which the antagonist AM281 was able to reverse the sedative 

effect of the agonist WIN55,212-2 [32]. The AM281 effectively restored 

the activity to normal levels (Fig. 10). In addition, AM281 alone was 

found to significantly stimulate locomotor activity between 1 and 2 h 

after its administration (Fig. 11). Both the antagonism of the effect of 

WIN55,212-2 and the effect of AM281 alone increased progressively with 

doses up to 0.3 mg/kg AM281, but did not further increase at 1 mg/kg. 

A 50% occupancy of the CB1 receptor, as assessed by inhibition of 

[ 131 I]AM281 binding, was achieved at a dose of 0.45 mg/kg. These data 

are consistent with prior in vitro indications that AM281 is a CB1 receptor 

antagonist or inverse agonist [33] and that AM281 inhibits an 


Figure 10 Effect of increasing doses of AM281 on binding of [ 131 I]AM281 in 

cerebellum and hippocampus, using brain stem as reference tissue. 

endogenous cannabinoid tone. This baseline activity of the CB1 system 

might be maintained either by constitutive activity of the receptor [34], or 

by endogenous agonists such as anandamide [11]. These experiments [32] 

indicate that PET could be used to measure the degree of occupancy of 

CB1 receptors by antagonist or inverse agonist drugs in the human brain, 

if these drugs turn out to have useful therapeutic effects, such as reducing 

memory loss in the elderly [35–37]. 

V. CONCLUSIONS 

Our development of [ 123 I]AM281, an antagonist radioligand for brain 

cannabinoid receptors, has allowed us to image this receptor for the first 

time in vivo. Ex vivo autoradiographic experiments have been conducted 

in rodents, and SPECT studies have been conducted in baboons. Research 


Figure 11 Stimulation of locomotor activity at CB1 receptor occupancy levels 

calculated from the effect of increasing doses of AM281 on binding of 

[ 131 I]AM281; squares, antagonism of the sedative effect of WIN55,212-2 at 0 

to 15 min; triangles, induction of hyperactivity by AM281 alone at 61 to 120 min. 

continues to develop superior radioligands for SPECT research and also 

to develop CB1 receptor radioligands that can be labeled with positronemitting 

nuclides for PET. The results of the animal work to date will 

provide the foundation for using AM281 and other cannabinoid receptor 

radioligands in human imaging experiments. It is predicted from our 

animal data that psychoactive or medicinal doses of agonists such as THC 

will not alter CB1 receptor radioligand binding in the human brain. On 

the other hand, PET or SPECT is likely to be useful in determining the 

degree of CB1 receptor occupancy necessary for therapeutic effects of 

antagonist drugs, as well as in evaluating CB1 receptor changes in 

addiction and in other diseases. 


This research was carried out at the Brookhaven National Laboratory 

under contract DE-AC02-98CH10886 with the U.S. Department of 


Energy and supported by its Office of Health and Environmental 

Research. The research was also supported by awards from the National 

Institute on Drug Abuse to AM (DA 07515, DA 09158) and to ANG 

(DA 12412). 

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6 

Structural and Pharmacological 

Aspects of Peptidomimetics 

Peter W. Schiller, Grazyna Weltrowska, Ralf Schmidt,* 

Thi M.-D. Nguyen, Irena Berezowska, Carole Lemieux, 

Ngoc Nga Chung, Katharine A. Carpenter,* and Brian C. Wilkes 

Clinical Research Institute of Montreal, Montreal, Quebec, Canada 


Small linear neuropeptides are structurally flexible molecules, capable of 

existing in a number of different conformations of comparably low energy. 

This structural flexibility precludes the determination of the bioactive 

conformation in solution and, furthermore, may be responsible for the 

lack of receptor selectivity of many of the naturally occurring peptide 

hormones and neurotransmitters, since conformational adaptation to 

different receptor topographies takes place. In recent years, the introduction 

of conformational constraints into peptides either locally or at a 

particular amino acid residue (N a or C a methylation, substitution of 

dehydro- or cyclic amino acids, etc.) or more globally (peptide cyclizations) 

emerged as a successful concept in the design of peptide analogues and 

peptidomimetics. In many cases, conformationally restricted peptide 

analogues showed high receptor selectivity and greatly improved conformational 

integrity. That different receptor types for a given peptide 

hormone or neurotransmitter differ from one another in their conforma- 

*Present address: AstraZeneca Research Centre Montreal, St. Laurent, Quebec, Canada. 


tional requirements toward the peptide ligand was first unambiguously 

demonstrated in the case of opioid receptors by comparing the receptor 

affinity profiles of a cyclic enkephalin analogue and its corresponding 

open-chain analogue [1]. Furthermore, conformationally restricted peptide 

analogues often showed enhanced stability against enzymatic degradation 

because a scissile peptide bond in the native peptide may no longer 

be susceptible to enzymatic cleavage when incorporated in a conformationally 

constrained structure. Conformational restriction of peptides 

represents a first step in the rational approach to develop peptidomimetics 

because conformationally constrained analogues may serve as relatively 

rigid templates for further structural modification aimed at removing some 

of the less attractive peptide structural features (e.g., peptide backbone 

replacements). In this chapter we illustrate these principles with examples 

from the opioid peptide field. 

The existence of three major classes (A, y, n) of opioid receptors is 

now well established [2,3]. Furthermore, the results of classical pharmacological 

testing and of opioid receptor binding studies indicate that various 

opioid receptor subtypes might also exist [4,5]. Recent work in the cloning 

of opioid receptors confirmed the existence of a y [6,7], a n [8], and a A [9] 

receptor, but so far has not led to the identification of receptor subtypes. 

Like all other G-protein-linked receptors, the three opioid receptors show 

seven putative transmembrane helices and considerable sequence similarity 

(60–70%) among themselves. Highest sequence identity is observed in 

the membrane-spanning segments and in the intracellular loops, whereas 

lower homology is seen in the extracellular regions. The ligand binding site 

is thought to be located in the cavity within the transmembrane domain of 

the receptors, whereas the extracellular loops may act as filters for the 

ligands, thus possibly playing a role in ligand selectivity. The precise threedimensional 

structures of the opioid receptors remain elusive because 

x-ray diffraction or NMR spectroscopic analysis cannot be used for their 

determination. However, theoretical analyses led to approximate models 

that are of interest for ligand docking studies and for the testing of 

hypotheses related to ligand design [10]. 

Since the discovery of the enkephalins in 1975 [11] a large number of 

endogenous opioid peptides have been detected in mammals, and at 

present three distinct families of opioid peptides are known (for a review, 

See Ref. 12). These are the enkephalins, the endorphins (a-, h-, and g-), and 

the dynorphins and neoendorphins. The recently discovered endomorphins 

[13] also may represent endogenous opioid peptides. Peptides with 

opioid activity have also been isolated from tryptic digests of milk casein 


(h-casomorphins) [14] and from frog skin—dermorphins [15] and deltorphins 

[16]. Like the morphine-related opiates, opioid peptides produce a 

large spectrum of central and peripheral effects, including analgesia, 

tolerance and physical dependence, respiratory depression, euphoria, 

dysphoria and hallucinations, sedation, feeding and other behavioral 

effects, hypothermia/hyperthermia, miosis, effects on tumor growth, control 

of release of several peptide hormones and catecholamines, effects on 

transit in the gut, and various cardiovascular effects. It has not yet been 

possible to establish clear-cut relationships linking specific opioid receptor 

types to distinct opioid effects. This is mainly because until recently, potent, 

stable agonists and antagonists with high specificity for the various 

receptor types have not been available. The results of several studies 

suggest that the A receptor plays a primary role in mediating analgesia; 

however, there is some evidence that y and n interactions may result in 

analgesic effects as well. Among the various isolated organ preparations 

used in the in vitro bioassays, the guinea pig ileum (GPI) contains primarily 

A receptors but also n receptors; in the mouse vas deferens (MVD) y 

receptors are predominant, even though A and n receptors are also present 

(for a review, see Ref. 17). It is now well recognized that the binding of 

agonists to opioid receptors leads to inhibition of adenylate cyclase via 

interaction with a guanine nucleotide regulatory protein [18]. 

The various endogenous opioid peptides resulting from processing of 

the three mammalian precursor molecules display only limited selectivity 

toward the different receptor types [12]. The only naturally occurring 

opioid peptides with high receptor specificity discovered so far are the yselective 

deltorphins [16] and, possibly, the endomorphins [13]. Medicinal 

chemists and peptide chemists have made numerous efforts to develop 

opioid agonists and antagonists with improved receptor selectivity. Substantial 

progress has been made in the development of selective nonpeptide 

opioid receptor ligands (for a review, see Ref. 19). The design of opioid 

peptide analogues with high receptor selectivity has also been very 

successful (for reviews, see Refs. 20 and 21). Structural modification of 

the enkephalins, h-casomorphin (morphiceptin), dermorphin, and the 

deltorphins through various amino acid and end group substitutions 

produced linear analogues that turned out to be highly selective A agonists, 

y agonists, or y antagonists. The preparation of numerous linear analogues 

of dynorphin A led to several potent n agonists with high n-receptor 

selectivity (for a review, see Ref. 22), whereas potent and selective n opioid 

antagonists structurally derived from dynorphin A have not yet been 

reported. A most successful strategy in opioid peptide analogue design 


has been the incorporation of conformational constraints through peptide 

cyclizations or substitution of conformationally restricted amino acids. In 

particular, conformational restriction of the enkephalins or the dermorphins 

and deltorphins produced potent and highly selective A agonists, y 

agonists and y antagonists. 

Agonists and antagonists showing high specificity for a particular 

opioid receptor class are valuable as pharmacological tools and may also 

have potential as therapeutic agents. Futhermore, it has been recognized 

that the development of opioid compounds with mixed agonist/antagonist 

properties may lead to improved analgesics with minimal side effects. In 

this chapter we discuss the development of highly selective A agonists and y 

antagonists, the first known compounds with mixed A agonist/y antagonist 

properties and a new class of dipeptide y agonists. 

II. M OPIOID AGONISTS 

A. Cyclic Peptides with M-Agonist Properties 

The first conformationally restricted opioid peptide reported in the 

literature was the cyclic enkephalin analogue H-Tyr-c[-D-A2bu-Gly-Phe- 

Leu-] (A2bu = a,g-diaminobutyric acid), which showed considerable 

preference for A receptors over y receptors [23]. Homologues of this 

compound containing a D-ornithine or D-lysine residue in place of D- 

A2bu also were A-selective agonists [24]. Analogues of H-Tyr-c[-D-Lys- 

Gly-Phe-Leu-] having one or two reversed amide bonds showed further 

improved A selectivity and excellent stability against enzymatic degradation 

[25]. One of the most selective cyclic opioid peptides with A agonist 

properties reported to date is the dermorphin-related tetrapeptide H-Tyr- 

D-O j 

rn-Phe-As j 

p-NH2 [26].Atheoreticalconformationalanalysisperformed 

with this compound revealed that the 13-membered peptide ring 

structure was highly constrained and that the lowest energy conformer was 

characterized by a tilted stacking interaction between the Tyr 1 and Phe 3 

aromatic rings [27]. Expansion of the peptide ring structure in this 

analogue, as achieved by replacement of Asp with Glu, resulted in the 

compound H-Tyr-D-O j 

rn-Phe-Glu j 

-NH2, which showed only slight preference 

for A receptors over y receptors [28]. The results of a molecular 

dynamics simulation carried out with this analogue showed that its 14membered 

peptide ring structure had moderate structural flexibility, while 

the exocyclic Tyr 1 residue and the Phe 3 side chain enjoyed considerable 

orientational freedom [29]. 


To limit the structural flexibility of these two aromatic residues, 

conformationally restricted analogues of Phe and/or Tyr were substituted 

[28,30] (Fig. 1). 

Replacement of Phe in the parent peptide with the cyclic phenylalanine 

analogue 2-aminoindan-2-carboxylic acid (Aic) resulted in a 

compound that showed only four times lower A affinity but 65 times lower 

affinity for y receptors and, consequently, markedly improved A selectivity 

j 

y A 

(K i/K i =49.6) (Table 1). The analog H-Tyr-D-Orn-Atc-Glu j 

-NH2, containing 

the conformationally constrained phenylalanine analogue 2-aminotetralin-2-carboxylic 

acid (Atc) at the 3 position of the peptide sequence, 

also retained high A-agonist potency and showed further improved A 

selectivity. Interestingly, the diastereomeric D-Atc 3 analogue also displayed 

good A receptor affinity and high A selectivity. This observation is 

in contrast to the weak affinity observed with the D-Phe 3 analogue in 

comparison to the L-Phe 3 parent peptide. Thus, stereospecificity was lost as 

a consequence of side chain conformational restriction, presumably 

because the D-Atc 3 analogue binds to the receptor in a manner different 

from that of the D-Phe 3 analogue. Replacement of Tyr 1 in the cyclic parent 

peptide with 6-hydroxy-2-amino-tetralin-2-carboxylic acid (Hat) produced 

the compound H-Hat-D-Or j 

n-Phe-Glu j 

-NH2, with only about three 

times reduced affinity for A and y receptors. Its diastereomer, H-D-Hat-D- 

Or j 

n-Phe-Glu j 

-NH2, was a full agonist at both the A and the y receptor but 

showed a substantial decrease in potency. Finally, the conformationally 

highly constrained analogue H-(D,L)-Hat-D-O j 

rn-Aic-Glu j 

-NH2 also 

showed high A-receptor affinity and marked A selectivity. This compound 

essentially contains only two freely rotatable bonds and represents the 

most rigid, rationally designed opioid peptidomimetic reported to date. 

Figure 1 Structural formulas of cyclic analogues of phenylalanine and tyrosine. 


Table 1 Opioid Receptor Affinities of Cyclic Dermorphin Analogues a 

Compound K i A (nM) K i y (nM) K i y /K i A 

H-Tyr-D-O j 

rn-Phe-Glu j 

-NH2 0.981 3.21 3.27 

H-Tyr-D-O j 

rn-D-Phe-Glu j 

-NH2 1660 14,000 8.43 

H-Tyr-D-O j 

rn-Phe-Glu j 

-NH2 4.21 209 49.6 

H-Tyr-D-O j 

rn-Atc-Glu j 

-NH2 8.26 1,570 190 

H-Tyr-D-O j 

rn-D-Atc-Glu j 

-NH2 26.3 3,510 133 

H-Hat-D-O j 

rn-Phe-Glu j 

-NH2 2.91 10.8 3.71 

H-D-Hat-D-O j 

rn-Phe-Glu j 

-NH2 54.2 74.7 1.38 

H-(D,L)-Hat-D-O j 

rn-Aic-Glu j 

-NH2 7.68 119 15.5 

a Displacement of [ 3 H]DAMGO (A-selective) and [ 3 H]DSLET (y-selective) from rat brain 

membrane binding sites. 

B. Conformational Study of H-Hat-D-O j 

rn-Aic-Glu j 

-NH2 The results of a molecular mechanics study indicated that the lowest 

energy conformation of H-Hat-D-O j 

rn-Aic-Glu j 

-NH2 is still characterized 

by a tilted stacking interaction of the aromatic rings of the residues in 

positions 1 and 3 of the peptide sequence (B. C. Wilkes and P. W. 

Schiller, unpublished results) (Fig. 2). It had been suggested that this 

tilted stacking arrangement of the two aromatic rings might represent a 

structural requirement for high A-receptor affinity of the tetrapeptide H- 

Tyr-D-O j 

rn-Phe-As j 

p-NH2 and structurally related cyclic dermorphin 

analogues [31]. An alternative model of the A-receptor-bound conformation 

based on conformational analysis of morphiceptin analogues is 

characterized by a larger distance (f10 A˚ ) between the Tyr 1 and Phe 3 

aromatic rings [32]. The A-selective morphiceptin analogue H-Tyr-Pro- 

Phe(NMe)-D-Pro-NH2 (PL017) in this proposed bioactive conformation 

is depicted in Figure 2. A conformer of H-Hat-D-O j 

rn-Aic-Glu j 

-NH2 with 

an energy 1.1 kcal/mol higher than that of the lowest energy structure 

showed good spatial overlap of its Tyr 1 and Phe 3 aromatic rings and 

N-terminal amino group with the corresponding moieties in morphiceptin 

in this proposed bioactive conformation, the root-mean-square deviation 

being 1.1 A˚ (Fig. 2). Thus, reasonable low energy conformers consistent 

with either one of the two proposed bioactive conformations can be 

assumed by H-Hat-D-O j 

rn-Aic-Glu j 

-NH2, and both models remain plausible 

candidate structures for the A-receptor pharmacophore. 


Figure 2 Spatial overlap of low energy conformers of H-Hat-D-O j 

rn-Aic-Glu j 

- 

NH2 (heavy lines) with proposed models of the A-receptor-bound conformation 

(light lines) based on conformational analysis of H-Tyr-D-O j 

rn-Phe-As j 

p-NH2 [24] 

(left panel) and H-Tyr-Pro-Phe(NMe)-D-Pro-NH2 (PL017) [29] (right panel). 

III. D OPIOID ANTAGONISTS 

The first y antagonists derived from opioid peptides were obtained through 

diallylation of the N-terminal amino group of enkephalin analogues. The 

best known compound of this type is N,N-diallyl-Tyr-Aib-Aib-Phe-Leu- 

OH (ICI 174,864; Aib = a-aminoisobutyric acid), which is quite y selective 

(K i A /K i y = 128) but not very potent (K i y = 199 nM; Ke =69nMinthe 

MVD assay) [33,34]. The nonpeptide y antagonist naltrindole (NTI) [35] is 

highly potent but displays only modest y selectivity (K i A /K i y = 21.2). A 

benzofuran analogue of naltrindole, NTB, showed improved y selectivity 

but somewhat lower y-antagonist potency [36]. However, both NTI and 

NTB also turned out to be antagonists against A and n agonists in the GPI 

assay, with potencies (Ke = 29–48 nM) about 100 to 300 times lower than 

those observed against y agonists in the MVD assay (Ke = 0.13 and 0.27 

nM, respectively) [36]. The recently discovered TIP(P) peptides represent a 

novel class of potent and highly selective y-opioid antagonists [37]. The two 

prototype antagonists were the tetrapeptide H-Tyr-Tic-Phe-Phe-OH 

(TIPP;Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) and the 


tripeptide H-Tyr-Tic-Phe-OH (TIP). TIPP showed high antagonist 

potency against various y agonists in the MVD assay (Ke = 3–5 nM), 

high y-receptor affinity (K i y = 1.22 nM) in the rat brain membrane receptor 

binding assay, and extraordinary y selectivity (K i A /K i y = 1410). Importantly, 

TIPP displayed no A-orn- antagonist properties in the GPI assay at 

concentrations as high as 10 AM. In comparison with TIPP, the tripeptide 

TIP was a somewhat less potent and less selective y antagonist. 

A. Structure–Activity Studies of TIP(P) Peptides 

Methylation of the N-terminal amino group of TIPP produced a compound, 

Tyr(NMe)-Tic-Phe-Phe-OH, with four fold enhanced y-antagonist 

potency and further improved y selectivity [38] (Table 2). Replacement of 

Tyr 1 in TIPP with 2V,6V-dimethyltyrosine (Dmt) led to the analog H-Dmt- 

Tic-Phe-Phe-OH (DIPP), with y-antagonist potency in the subnanomolar 

range (K e = 0.196 nM in the MVD assay) and with still excellent y 

selectivity. This compound turned out to be four times more potent than 

naltrindole as antagonist in the MVD assay (Table 2) and it represents the 

most potent y-opioid antagonist reported to date. The results of stability 

studies indicated that TIPP is stable in aqueous solution for extended 

periods of time but undergoes slow diketopiperazine formation and 

concomitant cleavage of the Tic 2 —Phe 3 peptide bond in DMSO or MeOH 

[39]. To prevent this spontaneous degradation, a TIPP analogue containing 

a reduced peptide bond between Tic 2 and Phe 3 was synthesized. The 

resulting pseudopeptide, H-Tyr-TicC[CH2–NH]Phe-Phe-OH (TIPP[C]), 

retained y-antagonist potency comparable to that of the parent peptide and 

showed extraordinary y selectivity in the receptor binding assays (K i A /K i y = 

10500) [40], being about 500 times more y selective than naltrindole and 17 

times more y selective than [D-Ala 2 ]deltorphin II (Table 2). Moreover, 

TIPP[C] was shown to be highly stable against chemical and enzymatic 

degradation. It also showed selectivity ratios exceeding 10,000 against all 

A- andn- receptor subtypes (A 1, A 2, n 1, n 2, n 3) [41] and thus represents an 

excellent pharmacological tool. The corresponding pseudotripeptide, 

H-Tyr-TicC[CH2-NH]Phe-OH (TIP[C]), also retained high y-antagonist 

potency, and its y selectivity was 40 times greater than that of its parent, 

TIP. Methylation of the secondary amino group of the reduced peptide 

bond in TIPP[C] produced the compound H-Tyr-TicC[CH2-NCH3]Phe- 

Phe-OH, which retained the high y antagonist potency of the parent 

pseudopeptide and showed even higher y selectivity (K i A /K i y = 15,900, 

Table 2). This compound is nearly 300 times more y selective than DPDPE 


Table 2 Antagonist Potencies and Opioid Receptor Affinities of TIPP Analogues 

Compound 

Ke 

(nM) a 

K i A 

(nM) b 

K i y 

(nM) b 

K i A /K i y 

H-Tyr-Tic-Phe-Phe-OH (TIPP) 5.86 1,720 1.22 1,410 

H-Tyr-Tic-Phe-OH (TIP) 11.7 1,280 9.07 141 

Tyr(NMe)-Tic-Phe-Phe-OH 1.22 13,400 1.29 10,400 

H-Dmt-Tic-Phe-Phe-OH 0.196 141 0.248 569 

H-Tyr-TicC[CH2-NH]Phe-Phe-OH 2.89 3,230 0.308 10,500 

(TIPP[C]) 

H-Tyr-TicC[CH2-NH]Phe-OH (TIP[C]) 9.06 10,800 1.94 5,570 

H-Tyr-TicC[CH2-NCH3]Phe-Phe-OH 4.76 13,400 0.842 15,900 

H-Tyr(3V-I)-Tic-Phe-Phe-OH Agonist 5,230 24.8 211 

H-Tyr(3V-I)-Tic-Phe-OH 141 12,100 60.0 202 

H-Tyr(3V-I)-TicC[CH2-NH]Phe-Phe-OH 19.2 2,660 2.08 1,280 

H-Tyr-Tic-Leu-Phe-OH 7.32 904 2.84 318 

H-Tyr-Tic-Ile-Phe-OH 12.7 6,460 4.37 1,480 

H-Tyr-Tic-Cha-Phe-OH (TICP) 0.438 3,600 0.611 5,890 

H-Tyr-TicC[CH2-NH]Cha-Phe-OH 0.219 1,050 0.259 4,050 

(TICP[C]) 

H-Tyr(3V-I)-Tic-Cha-Phe-OH 12.7 4,010 3.33 1,200 

H-Dmt-Tic-OH 6.55 1,360 1.84 739 

Naltrindole 0.636 3.86 0.182 21.2 

DPDPE Agonist 943 16.4 57.5 

[D-Ala 2 ]deltorphin II Agonist 3,930 6.43 611 

a Determined against DPDPE in the MVD assay. 

b Binding assay based on displacement of [ 3 H]DAMGO (A-selective) and [ 3 H]DSLET 

(y-selective) from rat brain membrane binding sites. 

and shows even slightly higher y-receptor selectivity than the TIPP[C] 

parent peptide. 

For the purpose of opioid receptor binding studies, TIPP was also 

radioiodinated. Surprisingly, [ 125 I]TIPP binding to y receptors in N4TG1 

neuroblastoma cells was substantially reduced in the presence of Na + and 

Gpp(NH)p [42]. These results indicated that substitution of an iodine atom 

at the 3V position of Tyr 1 in TIPP had turned the y antagonist into a y 

agonist. The corresponding ‘‘cold’’ analogue, H-Tyr(3’-I)-Tic-Phe-Phe- 

OH, was then synthesized and shown to be a full agonist in the MVD 

assay (IC50 = 97 nM). This agonist effect was antagonized by TIPP (Ke = 

11 nM) [38]. Corresponding iodination of the Tyr residue in TIP and 

TIPP[C] did not result in agonism, but somewhat reduced antagonist 


potency was observed (Table 2). It therefore appears that the astonishing 

conversion observed with the tetrapeptide TIPP may be due to an overall 

conformational effect rather than to a direct, local effect of the iodine 

substituent. Interestingly, substitution of a bromine or chlorine atom at the 

3V position of Tyr 1 in TIPP produced partial agonists with respective 

intrinsic efficacies of 0.16 and 0.12, whereas the Tyr(3V-F)-analogue was 

again a pure antagonist (K e = 13.0 nM) [38]. Thus, systematic substitution 

of halogen atoms beginning with iodine and in the order of the periodic 

table produced a progressive decrease in intrinsic activity and a concomitant 

increase in affinity at the y receptor (K i y = 24.2, 3.62, 3.00 and 1.62 

nM, respectively). 

Replacement of the Phe 3 residue in TIPP with the aliphatic amino 

acid residues Leu or Ile resulted in analogues that retained high yantagonist 

potency and considerable y selectivity (Table 2). This result is 

in agreement with the weak y-antagonist activity that had been reported for 

the tripeptide H-Tyr-Tic-Ala-OH [43]. Obviously, an aromatic residue at 

the 3 position of the peptide sequence of TIP(P) peptides is not absolutely 

required for y antagonist activity. Most interestingly, saturation of the 

Phe 3 aromatic ring in TIPP, as achieved through substitution of cyclohexylalanine 

(Cha), led to H-Tyr-Tic-Cha-Phe-OH [TICP], a compound 

showing substantially increased y-antagonist potency and higher y selectivity 

than the parent peptide [44]. The corresponding pseudopeptide, 

H-Tyr-TicC[CH 2-NH]Cha-Phe-OH (TICP[C]), showed a further 

improvement in y-antagonist activity. Its y-antagonist potency is comparable 

to that of the analogue H-Dmt-Tic-Phe-Phe-OH but, in comparison 

with the latter peptide, it is seven times more y selective (K i A /K i y = 4050) 

[44]. Both TIPP[C] and TICP[C] were prepared in tritiated form [45,46] 

and should turn out to be valuable new radioligands for y receptor labeling 

studies in vitro and in vivo. The analogue H-Tyr(3V-I)-Tic-Cha-Phe-OH 

was an antagonist in the MVD assay with a potency about 30 times lower 

than that of TICP. Thus, unlike in the case of TIPP, introduction of an 

iodine substituent at the 3V position of Tyr 1 in TICP did not produce a y 

agonist. This result demonstrates once again how a relatively subtle 

structural modification, such as the saturation of an aromatic ring, can 

have a determinant effect on agonist versus antagonist behavior. 

In 1995 the dipeptide H-Dmt-Tic-OH was reported to be a y-opioid 

antagonist with unprecedented y-receptor affinity (K i y = 0.022 nM) and y 

receptor selectivity (K i A /K i y = 150,000) [47]. However, in a direct comparison 

under identical assay conditions, this compound showed about 30 

times lower y-antagonist potency and 6 times lower y-receptor selectivity 


than TICP[C] [48] (Table 2). Similar results were obtained in a more recent 

study [49], which confirmed that H-Dmt-Tic-OH had much lower y 

receptor affinity [IC50(y) = 1.6 nM] and much lower y selectivity [IC50(A)/ 

IC50(y) = 558] than had originally been reported. Furthermore, H-Dmt- 

Tic-OH was found to be unstable in organic solvents owing to diketopiperazine 

formation (P. W. Schiller and T. M.-D. Nguyen, unpublished 

results). 

B. Conformational Studies of TIP and TIPP 

A molecular mechanics study (grid search and energy minimization) of the 

tripeptide y-antagonist TIP resulted in several low energy conformers 

having energies within about 2 kcal/mol of that of the lowest energy 

structure [50]. The centrally located Tic residue imposes a number of 

conformational constraints on the N-terminal dipeptide segment; however, 

the results of molecular dynamics simulations indicate that this 

tripeptide still shows some structural flexibility at the Phe 3 residue. 

Attempts to demonstrate spatial overlap between the pharmacophoric 

moieties of low-energy conformers of TIP and the structurally rigid nonpeptide 

y antagonist naltrindole were made by superimposing either the 

Tyr 1 and Phe 3 aromatic rings and the N-terminal amino group or the Tyr 1 

and Tic 2 aromatic rings and the N-terminal amino group of the peptide 

with the corresponding aromatic rings and nitrogen atom in the alkaloid 

structure. In each case the investigators found a conformer of TIP with an 

energy very close to that of the lowest energy structure (2.1 kcal/mol 

higher). However, the low-energy conformer showing spatial overlap of its 

Tic 2 aromatic ring with the six-membered aromatic ring of the indole 

moiety in naltrindole (Fig. 3) appears to be a more plausible candidate 

structure of the y-receptor-bound conformation for two reasons: 

1. The Tic 2 aromatic ring has been shown to be of crucial 

importance for y antagonist activity [51]. 

2. The y-antagonist properties are maintained upon replacement of 

the Phe 3 residue in the peptide with an aliphatic amino acid 

residue (see earlier). 

This model of the receptor-bound conformation of TIP is characterized by 

a clustered configuration of the three aromatic moieties with the Phe 3 

aromatic ring sandwiched between the Tyr 1 and Tic 2 aromatic rings. 

A molecular mechanics study of TIPP and TIPP[C] produced about 

70 structures within 3 kcal/mol of the lowest energy conformation in each 


Figure 3 Superimposition of a low energy conformer of TIP (heavy lines) with 

the minimized structure of naltrindole (light lines). The Tyr 1 and Tic 2 aromatic 

rings and the N-terminal amino group of the peptide are superimposed with the 

corresponding moieties in the alkaloid structure. The superimposed molecules 

are shown in two different orientations. 

case [52]. The lowest energy conformers of both TIPP and TIPP[C] 

showed good overlap of their Tyr 1 and Tic 2 aromatic rings and N-terminal 

amino group with the corresponding pharmacophoric moieties of naltrindole. 

Thus, these results are in agreement with the model of the 

receptor-bound conformation of TIP proposed earlier. This model is 

characterized by all-trans peptide bonds and was definitely confirmed by 

conformational analyses of two TIPP analogues (y antagonists) in which a 

cis peptide bond between the Tyr 1 and Tic 2 residues is sterically forbidden 

[53]. Both TIPP and TIPP[C] are very hydrophobic peptides, and the 

results of the theoretical conformational analyses clearly indicated that 

they enjoy considerable structural flexibility, particularly in their Cterminal 

dipeptide segment. There is no doubt that their conformations 

are quite dependent on the environment. According to our theoretical 

analysis, a crystal structure of TIPP published in 1994 [54] is about 3 kcal/ 

mol higher in energy than the lowest energy structure and shows no 

similarity to any of the calculated low energy structures [52,53]. The 

crystal structure of TIPP appears to be stabilized by a large number of 

intermolecular hydrophobic contacts between layers of TIPP molecules in 

the crystal and by several hydrogen bonds to solvent (AcOH) molecules. 

There is no reason to believe that it resembles the y-receptor-bound 

conformation of TIPP. In an aqueous environment TIP(P) peptides 

may undergo a so-called hydrophobic collapse [55]. It is possible that 

subtle structural modifications, such as introduction of an iodine sub- 


stituent at the 3V position of Tyr 1 , saturation of the Phe 3 aromatic ring, or 

reduction of the Tic 2 —Phe 3 peptide bond, may produce different patterns 

of aromatic ring clustering that could result in either y-agonist or yantagonist 

activity, as described earlier. 

C. Effect of D-Opioid Antagonists on the Development 

of Morphine Tolerance and Dependence 

Blockade of y receptors with the nonpeptide y antagonist naltrindole 

concurrently with chronic morphine treatment has been reported to 

attenuate the development of tolerance and the severity of the precipitated 

withdrawal syndrome in mice [56]. In an effort to corroborate these 

results, the effects of TIPP, TIPP[C], and naltrindole on the development 

of morphine tolerance and dependence were examined. Each of the 

antagonists was continuously infused into the lateral ventricle of rats 

treated chronically with subcutaneous morphine [57]. After a 6-day period 

of drug administration, rats treated with TIPP[C] showed no morphine 

tolerance and a greatly reduced incidence of withdrawal symptoms 

following injection of naloxone. Naltrindole and TIPP also significantly 

decreased the amount of time spent in withdrawal but did not attenuate 

the development of morphine tolerance. More recently, morphine was 

shown to retain its A-receptor-mediated analgesic activity in y-opioid 

receptor knockout mice without producing analgesic tolerance upon 

chronic administration [58]. These interesting findings clearly demonstrate 

that y-opioid receptors play a major role in the development of morphine 

tolerance and dependence and suggest the possibility of the combined use 

of a A type opioid analgesic and a y-opioid antagonist in the treatment of 

chronic pain. Even more interestingly, these results indicate that mixed A 

agonist/y antagonists can be expected to be analgesics with low propensity 

to produce tolerance and dependence and, therefore, might be of benefit in 

the management of chronic pain. 

IV. MIXED M AGONIST/D ANTAGONISTS 

A. Prototypes and Structure–Activity Relationships 

The first known example of a mixed A agonist/y antagonist was a TIPP 

analogue in which the free C-terminal carboxylate function had been replaced 

by a carboxamide function [37]. This compound, H-Tyr-Tic-Phe- 


Phe-NH2 (TIPP-NH2), was found to be a moderately potent A agonist in 

the GPI assay and a potent y antagonist in the MVD assay (Table 3). 

Replacement of Tyr 1 in TIPP-NH2 with Dmt produced the compound 

H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2), showing an increase in both Aagonist 

potency and y-antagonist activity by nearly two orders of magnitude 

[59,60]. In the receptor binding assays DIPP-NH2 displayed very high 

y-receptor affinity and still some preference for y receptors over A receptors. 

In comparison with DIPP-NH 2, the corresponding analogue with a reduced 

peptide bond between the Tic 2 and Phe 3 residues, H-Dmt-TicC 

[CH2-NH]Phe-Phe-NH2 (DIPP-NH2[C]), was about twice as potent as 

agonist in the GPI assay and about half as potent as antagonist in the 

MVD assay. Showing A- and y-receptor affinities that were both in the 

subnanomolar range, DIPP-NH2[C] was essentially nonselective (K i A /K i y 

= 2.11; Table 3). Therefore, DIPP-NH2[C] represents the first known 

opioid compound with balanced A-agonist/y-antagonist properties 

[59,60]. In the rat tail flick test, DIPP-NH 2[C] given intracerebroventricularly 

(ICV) produced a potent analgesic effect, being about three 

times more potent than morphine. It produced less acute tolerance than 

morphine, but still a certain level of chronic tolerance. Unlike morphine, 

DIPP-NH2[C] produced no physical dependence upon chronic administration 

at high doses. Thus, DIPP-NH2[C] fulfilled to a large extent 

the expectations based on the mixed A-agonist/y-antagonist concept [60]. 

Surprisingly, elimination of the C-terminal carboxylate function 

of the tripeptide y antagonist TIP resulted in H-Tyr-Tic-NH-(CH 2) 2-Ph 

(Ph = phenyl), a compound that was a moderately potent full y agonist 

(Table 3) [61]. Interestingly, lengthening of the phenylethyl substituent by 

insertion of an additional methylene group restored y antagonism, as 

indicated by the finding that the dipeptide derivative H-Tyr-Tic-NH- 

(CH2)3-Ph was a moderately potent y antagonist in the MVD assay and a 

relatively weak partial A agonist in the GPI assay. This remarkable 

dependence of y-agonist versus y-antagonist behavior on the length of 

the phenylalkyl substituent may be due to conformational effects resulting 

in different clustering of the three aromatic moieties present in these 

molecules. The analogue H-Dmt-Tic-NH-(CH2)3-Ph showed very high 

affinity for both A and y receptors and was a potent y antagonist in the 

MVD assay (Ke = 1.69 nM). That this compound displayed relatively 

modest agonist potency in the GPI assay suggests that it also may have 

partial A-agonist properties and that it may represent a mixed partial A 

agonist/y antagonist. In the case of high affinity A-receptor ligands, partial 

agonism is not always directly apparent in the GPI assay because the 


Table 3 In Vitro Opioid Activities and Receptor Affinities of Mixed A Agonist/y Antagonists 

GPI MVD Binding assays a 

Compound IC50, nM IC50, (nM) Ke (nM) b 

K i A (nM) K i y (nM) K i A /K i y 

H-Tyr-Tic-Phe-Phe-NH2 1700 18.0 78.8 3.00 26.3 

H-Dmt-Tic-Phe-Phe-NH2 18.2 0.209 1.19 0.118 10.1 

H-Dmt-TicC[CH2-NH]Phe-Phe-NH2 7.71 0.537 0.943 0.447 2.11 

H-Tyr-Tic-NH-(CH2)2-Ph 3010 82.0 69.1 5.22 13.2 

H-Tyr-Tic-NH-(CH2)3-Ph (42 %) c 

41.9 160 3.01 53.2 

H-Dmt-Tic-NH-(CH 2) 3-Ph 102 1.69 0.386 0.0871 4.43 

H-Tyr-c[-D-Orn-Phe-D-Pro-Gly-] 2.14 4.89 0.881 13.2 0.0667 

H-Tyr-c[-D-Orn-2-Nal-D-Pro-Gly-] 384 233 5.89 17.2 0.342 

H-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-] 7.88 2.13 0.460 0.457 1.01 

a Displacement of [ 3 H]DAMGO (A-selective) and [ 3 H]DSLET (y-selective) from rat brain membrane binding sites. 

b Determined against DPDPE. 

c Maximal inhibition of the contractions at 10 AM. 


ileum has a very high A-receptor reserve. Substituted Tyr-Tic-dipeptide 

amides with mixed A-agonist/y-antagonist properties are of interest because 

their small molecular size and lipophilic character may facilitate their 

passage across the blood–brain barrier (BBB). Further efforts aimed at 

strengthening the A-agonist component of this class of compounds may 

be required. 

Another prototype of a mixed A agonist/y antagonist is the cyclic hcasomorphin 

analogue H-Tyr-c[-D-Orn-2-Nal-D-Pro-Gly-] [62]. This 

compound turned out to be a fairly potent A agonist in the GPI assay 

and showed relatively modest y-antagonist potency in the MVD assay 

(Table 3). The 2-naphthylalanine (2-Nal) residue in this compound is a 

key structural determinant for its y-antagonist behavior, since the corresponding 

Phe 3 analogue, H-Tyr-c[-D-Orn-Phe-D-Pro-Gly-] was found to 

be a full y agonist in the MVD assay [62]. As expected, an analogue 

containing Dmt in place of Tyr 1 , H-Dmt-c-[-D-Orn-2-Nal-D-Pro-Gly-], 

showed greatly increased A-agonist and y-antagonist potency [63]. This 

pentapeptide displayed almost equal affinities for A and y receptors in the 

subnanomolar range and, thus, represents another example of a balanced 

A agonist/y antagonist. In comparison with DIPP-NH2[C], H-Dmt-c[-D- 

Orn-2-Nal-D-Pro-Gly-] has the same A-agonist potency in the GPI assay 

and is about four times less potent as a y antagonist in the MVD assay. 

The various compounds described in this section represent the only 

known mixed A-agonist/y-antagonist substances reported to date. Analgesic 

testing of all these prototypes will reveal which type of compound 

has the greatest potential for the development of viable analgesics. 

Further analogues may have to be prepared and examined to determine 

the ratio between A-agonist and y-antagonist potency required for 

optimal attenuation of tolerance and dependence development. Additional 

structural modifications may be necessary to increase analgesic 

potency and bioavailability. 

B. Conformational Study of H-Tyr-c[-D-Orn-2-Nal-D- 

Pro-Gly-] 

The conformation of the mixed A agonist/y antagonist H-Tyr-c[-D-Orn-2- 

Nal-D-Pro-Gly-] in comparison to that of H-Tyr-c[-D-Orn-Phe-D-Pro-Gly-] 

was studied in DMSO-d6 by NMR spectroscopy and by molecular mechanics 

calculations [62,64]. Neither peptide showed nuclear Overhauser 

effects between C a H protons or chemical exchange cross peaks in spectra 

obtained by total correlation and rotating frame Overhauser enhance- 


ment spectroscopy (TOCSY, ROESY). These results indicated that the 

average preferred solution conformation of both peptides was characterized 

by all-trans peptide bonds. The results of temperature-dependence 

studies of the amide proton chemical shifts in conjunction with those of 

the molecular mechanics studies indicated that the two analogues had 

backbone conformations that were both stabilized by Tyr 1 -CO HN- 

Phe 3 (or 2-Nal 3 )and D-Orn 2 -CO HN y -D-Orn 2 hydrogen bonds. Furthermore, 

ROESY experiments revealed a close proximity between the 

aromatic moiety of the 3-position residue and the pyrrolidine ring of the 

D-Pro 4 residue in these two compounds. The comparison of all calculated 

low-energy conformations with the various proton NMR parameters led 

to proposals for the solution conformation of these two peptides (Fig. 4). 

Inspection of the structures reveals that the Phe 3 - and 2-Nal 3 - 

analogues have similar backbone conformations and the same side chain 

orientation at the 3 position. These results suggest that the y-antagonist 

Figure 4 Proposed solution conformations of H-Tyr-c-[-D-Orn-2-Nal-D-Pro- 

Gly-] (left panel) and H-Tyr-c-[D-Orn-Phe-D-Pro-Gly-] (right panel). 


properties of the 2-Nal 3 analogue may not be due to a difference in its 

overall conformation in comparison to the Phe 3 analogue but rather may 

be the result of a direct interference of the 2-naphthyl moiety per se at the 

receptor binding site, preventing proper alignment of the peptide such as 

required for signal transduction. 

V. D AGONISTS 

y-Opioid agonists are known to produce analgesic effects and look 

promising because they induce less tolerance and physical dependence 

than morphine, no respiratory depression, and few or no adverse 

gastrointestinal effects [65,66]. Selective peptide y agonists currently 

available include the enkephalin analogues H-Tyr-D-Thr-Gly-Phe-Leu- 

Thr-OH (DTLET), H-Tyr-c[D-Pen-Gly-Phe-D-Pen]OH (DPDPE), and 

H-Tyr-c[D-Cys-Phe-D-Pen-OH]OH (JOM-13), as well a the deltorphins 

H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (dermenkephalin), H-Tyr-D- 

Ala-Phe-Asp-Val-Val-Gly-NH2 (deltorphin I), and H-Tyr-D-Ala-Phe- 

Glu-Val-Val-Gly-NH2 (deltorphin II) (for reviews, see Refs. 20 and 21). 

However, these peptides are of relatively large molecular size and for this 

reason their ability to cross the BBB is very limited. Nonpeptide y agonists 

that were developed in the early to mid-1990s include the racemic compound 

BW373U86 [67] and its chemically modified enantiomer SNC80 

[68], as well as the compound TAN-67 [69]. However, BW373U86 produced 

significant toxicity, manifested behaviorally as convulsions and 

barrel rolling, in mice [70], and TAN-67 showed no significant antinociceptive 

activity in the mouse tail flick test [69]. Evidently, there is still a need 

for the development of new potent y opioid agonists of low molecular 

weight and high lipophilicity. 

In an effort to increase the moderate y-agonist potency and the yreceptor 

selectivity of the dipeptide H-Tyr-Tic-NH-(CH 2) 2-Ph [61], structural 

modifications of the C-terminal phenylethyl group were performed 

by introduction of an additional substituent either in ortho position of the 

phenyl ring or at the h carbon [44] (Table 4). The analogue H-Tyr-Tic-NH- 

(CH2)2-Ph(o-Cl) was a 10-fold more potent y agonist than the parent 

peptide in the MVD assay and was five times more y-receptor selective. 

Introduction of a second phenyl group at the h carbon of the phenylethylamine 

moiety led to the compound H-Tyr-Tic-NH-CH2-CH(Ph)2, with 20fold 

increased y-agonist potency and 2-fold improved y selectivity. The 

corresponding N-methylated analogue, Tyr(NMe)-Tic-NH-CH 2-CH(Ph) 2 


Table 4 In Vitro Opioid Activities of Dipeptide y-Opioid Agonists 

Compound 

displayed subnanomolar y-agonist potency and still marked y-receptor 

selectivity. One of the isomers (I) of H-Tyr-Tic-NH-CH2-CH(Ph)COOEt 

was also found to be a potent y agonist with very high preference for y 

receptors over A receptors. An analogue containing 2V-hydroxy,6V-methyltyrosine 

(Hmt) in place of Tyr 1 , H-Hmt-Tic-NH-CH 2-CH(Ph) 2, turned 

out to be particularly remarkable because it showed both subnanomolar y 

agonist potency (IC 50 = 0.630 nM) and very high y-receptor selectivity 

(K i A /K i y = 835). In a direct comparison under identical assay conditions, 

this compound was 8 times more potent than the well-known y agonist 

DPDPE and 15 times more y selective. None of these compounds had 

significant binding affinity for n-opioid receptors. From these results it can 

be concluded that the dipeptide derivatives described here represent a new 

class of potent and selective y-opioid agonists. It is expected that these 

compounds should be able to cross the BBB to some extent because of their 

low molecular weight and high lipophilicity. Therefore, they have potential 

as centrally acting analgesics that may produce fewer side effects than the 

currently used A type opiates. 

VI. CONCLUSIONS 

IC 50 

(nM) a 

K i y 

(nM) b 

K i A 

(nM) b 

K i A /K i y 

H-Tyr-Tic-NH-(CH2)2-Ph 82.0 5.22 69.1 13.2 

H-Tyr-Tic-NH-(CH2)2-Ph(o-Cl) 8.77 1.43 96.9 67.8 

H-Tyr-Tic-NH-CH 2-CH(Ph) 2 3.77 0.981 28.8 29.4 

Tyr(NMe)-Tic-NH-CH 2-CH(Ph) 2 0.261 0.581 12.7 21.9 

H-Tyr-Tic-NH-CH2-CH(Ph)COOEt (I) 1.28 0.569 886 1560 

H-Tyr-Tic-NH-CH2-CH(Ph)COOEt (II) 8.64 3.03 153 50.5 

H-Hmt-Tic-NH-CH 2-CH(Ph) 2 0.630 2.00 1670 835 

DPDPE 5.30 16.4 943 57.5 

a Determined in the MVD assay. 

b Binding assay based on displacement of [ 3 H]DSLET (y-selective) and [ 3 H]DAMGO 

(A-selective) from rat brain membrane binding sites. 

Application of the concept of conformational restriction to opioid peptides 

has produced fruitful results, insofar as peptide analogues and mimetics 

with interesting opioid activity profiles and high stability against enzymatic 


degradation were obtained. The conformationally restricted analogues 

that were developed were amenable to meaningful conformational analysis 

permitting the elaboration of models of the bioactive conformation at the A 

or y receptor. 

The multiple conformational restriction of dermorphin-related tetrapeptide 

analogues that was performed represents a rational design of 

opioid peptidomimetics characterized by a high degree of structural rigidification. 

This is indicated by the fact that the A-selective agonist H-Hat-D- 

O j 

rn-Aic-Glu j 

-NH2 contains only two freely rotatable bonds, whereas there 

are 14 freely rotatable bonds in [Leu 5 ]enkephalin. 

The discovery of the TIP(P) peptides and their further structural 

modification led to y opioid antagonists with unprecedented potency and 

selectivity. The observation that very subtle structural modifications of 

these flexible and hydrophobic peptides in some cases converted a y 

antagonist into a y agonist and vice versa is most intriguing and unique 

in the peptide field. This behavior may be explained with changes in the 

patterns of aromatic ring clustering in these hydrophobically collapsed 

molecules as a consequence of the minor structural alterations (introduction 

of a halogen substituent, peptide bond reduction, saturation of an 

aromatic ring, etc.) that were performed. The TIP(P) peptides are of 

therapeutic interest because y antagonists have been shown to attenuate 

the development of morphine tolerance and dependence [56,57] and to 

have an immunosuppressive effect [71]. 

The three prototype mixed A agonist/y antagonists described in this 

chapter have excellent potential as analgesics with low propensity to 

produce tolerance and dependence. The pseudotetrapeptide DIPP- 

NH2[C] has already been shown to produce a potent analgesic effect, less 

tolerance than morphine, and no physical dependence upon chronic 

administration. In preliminary experiments, the tetrapeptides DIPP-NH2 

and DIPP-NH2[C] were shown to cross the BBB to some extent, but 

further structural modifications need to be performed in order to improve 

the BBB penetration of these compounds. The Tyr-Tic dipeptide derivatives 

can also be expected to penetrate into the central nervous system 

because they are relatively small, lipophilic molecules. In this context, it is 

of interest to point out that the structurally related dipeptide H-Dmt-D- 

Ala-NH-(CH2)3-Ph (SC-39566), a plain A-opioid agonist, produced antinociception 

in the rat by subcutaneous and oral administration [72]. As 

indicated by the results of the NMR and molecular mechanics studies, the 

conformation of the cyclic h-casomorphin analogue H-Tyr-c[-D-Orn-2- 

Nal-D-Pro-Gly-] is stabilized by intramolecular hydrogen bonds. There- 


fore, this mixed A agonist/y antagonist has a reduced capacity for intermolecular 

hydrogen bonding with water molecules and, consequently, 

should have a reasonable chance to cross the BBB as well. 

The dipeptide y agonists may turn out to be interesting pharmacological 

tools, since some of them are more potent and more selective than 

the y agonists currently in use. Furthermore, these compounds represent a 

new class of y agonists and have potential for pain treatment because they 

may also be small enough and lipophilic enough to cross the BBB and to 

produce a centrally mediated analgesic effect. 


The work described in this chapter was supported by operating grants 

from the Medical Research Council of Canada (MT-5655) and the National 

Institute on Drug Abuse (DA-04443). 

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40. Schiller PW, Weltrowska G, Nguyen TM-D, Wilkes BC, Chung NN, 

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receptor antagonist with extraordinary y selectivity. J Med Chem 1993; 

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41. Visconti, L.M., Standifer, K.M., Schiller, P.W., Pasternak, G.W. TIPP[C]: a 

highly selective y ligand. Neurosci Lett 1994; 181:47–49. 

42. Lee PHK, Nguyen TM-D, Chung NN, Schiller PW, Chang KJ. Tyrosineiodination 

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stable delta opioid antagonist. Life Sci (Pharmacol Lett) 1995; 56:PL225– 

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7 

Linkers and Resins for Solid-Phase 

Synthesis 

Pan Li and Elaine K. Kolaczkowski 

Vertex Pharmaceuticals, Inc., Cambridge, Massachusetts, U.S.A. 

Steven A. Kates 

Surface Logix, Inc., Brighton, Massachusetts, U.S.A. 


Organic chemistry in the last half of the twentieth century has evolved to a 

level of extreme sophistication in which complex macromolecules thought 

only to exist in nature were prepared in a laboratory hood. The process 

typically involves performing a reaction in an organic solvent followed by 

isolating, purifying, and analyzing the compound. This tedious, timeconsuming 

procedure requires considerable expertise. Bruce Merrifield 

was the first to recognize an alternative approach for the preparation of 

organic compounds. He applied this method to synthetic peptides and was 

awarded the Nobel Prize in 1984 for this discovery [1]. The concept was to 

perform the chemistry proven in solution but add a covalent attachment 

step that links the target to an insoluble polymeric support. Key advantages 

to the solid-phase technique include simple filtration, washing without 

manipulative losses, and ease of automation. 

Peptide synthesis was amenable to solid-phase techniques since the 

process was repetitive. The C-terminal amino acid is attached to polymeric 

surface and the peptide chain is assembled via a two-step process: coupling 

of the incoming amino acid that has the alpha-amino group protected 


and removal of this protecting group. Following chain elongation, the 

peptide is liberated from the solid support with concomitant release of the 

side-chain-protecting groups. Subsequent to Merrifield’s discovery, other 

repetitive processes such as DNA synthesis and protein sequencing were 

adapted to solid-phase methods. 

Most peptides occur in nature as C-terminal acids or amides and 

methods were developed to release a peptide from a solid support to 

provide these two chemical functionalities. During the 1980s, research in 

peptide synthesis methods focused on releasing peptides from a polymeric 

support using various conditions (high acid concentration, low acid 

concentration, basic, etc.). During the early 1990s, there was a realization 

that solid-phase techniques could be applied to the construction of small 

molecules for drug discovery. Since many drugs do not contain carboxylic 

acids or amides, there was a need to expand the resulting chemical 

functionality at the anchoring position of the molecule following cleavage 

from solid support. Thus there has been a recent resurgence in research for 

solid-phase methods. 

A critical aspect to solid-phase synthesis is the anchoring of the 

molecule to the polymeric support. A solid support or resin is required to 

possess a functional group that is the starting point for the construction of 

the molecule. In addition, resins should possess the following properties: 

(1) mechanically robust; (2) stable to temperature variation; (3) good 

swelling in a broad range of solvents; (4) acceptable bead size and loadings; 

(5) stable with acidic, basic, reducing, and oxidizing conditions; (6) 

compatible with radical, carbene, carbanion, and carbenium ion chemistry; 

(7) biocompatible and swelling in aqueous buffers; (8) little nonspecific 

binding to biomolecules; (9) mobile, well-solvated, and reagent-accessible 

sites. The two most commonly used supports are polystyrene (PS) functionalized 

with a chloromethyl 1 (original Merrifield polymer) or amino 

group 2 at the terminus (Fig. 1) [2]. For peptide synthesis, the cesium salt of 

a protected amino acid is anchored to chloromethyl-PS via a nucleophilic 

displacement of chlorine. Following chain elongation, the peptide is 

released from the support by treatment with a strong acid such as HF to 

provide a peptide acid. Amino acids anchored to aminomethyl-PS form an 

Figure 1 


Figure 2 

amide bond and are stable to HF conditions. p-Methylbenzhydrylamine 

(MBHA, 3) is an amine functionalized polystyrene that liberates Cterminal 

amides upon exposure to HF (Fig. 2) [3]. 

A second strategy is to attach a linker (also referred to as a handle or 

anchor) to the resin followed by assembly of the molecule. A linker is 

bifunctional spacer that serves to link the initial synthetic unit to the 

support in two discrete steps (Fig. 3). To attach a linker to a chloromethyl- 

PS resin, a phenol functionality such as handle 4 is used to form an ether 

bond (Fig. 4). To attach the same handle to an amino-functionalized 

support, acetoxy function 5 or a longer methylene spacer of the corresponding 

phenol is applied to form an amide bond. Both of these resins 

perform similarly and only differ in their initial starting resin [4]. An 

alternative approach is to prepare a preformed handle in which the first 

building block is prederivatized to the linker and this moiety is attached to 

the resin. For peptide synthesis, this practice is common for the preparation 

of C-terminal peptide acids in order to reduce the amount of 

racemization of the a-carbon at the anchoring position [5]. 

There are three features of a linker that will determine which support 

is applicable to a synthetic scheme: (1) the functionality of the molecule at 

the anchoring position required for attachment; (2) cleavage conditions; 

and (3) the resulting functionality at the anchoring position of the molecule 

after the cleavage. As a continuing review on resins and linkers, this 

discussion will focus on the work that has been developed from 1997 to 

1999 (Refs. 6–9 and references cited therein) and is described according to 

Figure 3 


Figure 4 

the resulting functionality at the anchoring position after the cleavage. In 

addition, references and structures will be provided herein for the key 

linkers/resins that are commercially available or routinely used in solidphase 

peptide and organic synthesis. 

II. RESINS AND LINKERS FOR CARBOXYLIC 

ACID GENERATION 

Most peptides contain a carboxylic acid or carboxamide at the Cterminus 

of the polymer chain. Since Merrifield introduced solid-phase 

peptide synthesis in 1963, peptide chemists developed linkers that will 

yield these two functionalities upon release from the solid support using a 

variety of chemical conditions. The hydroxyl-containing resins (Fig. 5) 

based upon alkoxy-substituted benzyl alcohols were developed to supplement 

chloromethyl- and hydroxymethyl-PS. Carboxylic acids are esterified 

to the resin typically using N,NV-diisopropylcarbodiimide (DIPCDI). 

The acid strength required for release of the molecule from the solid 

support is related to the electron donor substituents on the benzene ring 

which stabilize the transient resin-bound carbocation. With greater 

resonance stabilization conveyed by additional alkoxy groups and aryl 

rings, milder acidic conditions are required for cleavage. PAM ( phydroxymethylphenylacetic 

acid) resin 6 [10] with no electron-donating 

groups requires HF treatment while Wang 7 [11], HMPA 8 (4-hydroxymethylphenylacetic 

acid, also referred to as PAB [ p-alkoxylbenzyl] or 

PAC [peptide acid]) [12] and DHPP 9 (4-(1V,1V-dimethyl-1V-hydroxypropyl)phenoxyacetyl) 

[13] contain resonance stabilizing groups and cleavage 

is affected with trifluoroacetic acid (TFA). The hydroxymethylbenzyl 


Figure 5 

linker 10 (HMB) [14] contains a carbonyl group para to the ester anchor 

and is activated to nucleophilic attack such as hydroxide ion and is stable 

toward acid. Alkoxybenzyl derivatives with greater electron donor 

strength (Fig. 6) such as SASRIN (super-acid-sensitive resin) 11 [15], 

Rink acid 12 [16], and HAL (hyper-acid sensitive) 13 [17] resin allow 

carboxylic acids to be cleaved using a lower acid concentration (typically 

Figure 6 


Figure 7 

1–10% TFA) in DCM (dichloromethane). Linkers for carboxylic acids 

have also been designed to effect cleavage via photolysis (3-nitro-4hydroxymethylbenzoic 

acid, ONb 14) [18] and flouridolysis (N-3 or 4) 

((4-hydroxymethyl)-phenoxy-t-butylphenylsilyl)phenyl pentanedioic acid 

monoamide (PBs) 15 [19] and quinonemethide-based handle 16 [20] (Fig. 

7). 

Fluorenone derived linker 17 prepared in two steps was coupled to 

aminomethyl-PS via DIPCDI [21]. Due to the presence of an electronwithdrawing 

carboxamide group, the release of carboxylic acids from this 

support requires strong acids, such as trifluoromethanesulfonic acid 

(TFMSA) (Scheme 1). Insertion of an oxygen adjacent to the biphenyl 

rings to the fluorenone scaffold provides xanthene 18 handle [22]. The 

oxygen is strategically located to decrease the acid concentration required 

Scheme 1 


Scheme 2 

for cleavage and carboxylic acids are released using TFA (Scheme 2). Resin 

bound diazo linker 19 was synthesized starting from Wang resin and was 

further oxidized to a benzyl aldehyde (Scheme 3) [23]. Carboxylic acids are 

anchored to the support in a rapid, colorimetric reaction and are released 

upon TFA treatment. 

Photolabile linkers play an important role in solid-phase organic 

synthesis (SPOS) due to their stability under both acidic and basic 

conditions. The ONb photolabile linker was modified to improve cleavage 

rates and yields; Fmoc-Tos-OH was released in 87% yield after 23 h 

(Scheme 4) [24]. Specifically, the primary alcohol was changed to a 

secondary benzylic alcohol and the attachment to the resin was through 

an alkyl chain as opposed to an amide function. Linker 20 was used for 

the production of carboxylic acids or carbohydrates. A second example 

Scheme 3 


Scheme 4 

incorporated a dithiane function to serve as a safety catch against 

premature photoreaction [25–26]. A carboxylic acid functionality was 

coupled to linker 21 via DIPCDI, the dithiane protecting group was 

removed by an S-alkylating reagent such as methyl triflate, and release of 

the molecule was accomplished with UV irradiation in THF-methanol 

(Scheme 5). Based on 2-pivaloylglycerol, photolabile linker 22 was 

prepared in six steps from the dimer of 1,3-dihydroxyacetone (Scheme 

6) [27]. The handle was attached to TentaGel S NH2 amino resin, the 

protecting groups from the hydroxyl functions were removed, and a series 

of peptides were assembled. Cleavage rates were reported to be faster 

than other photolabile linkers. 

Silyl-based linker 23, cleaved by either basic (TBAF) or neutral 

(CsF) fluoridolysis to release carboxylic acids, alcohols, or amines, was 

prepared by treatment of a Grignard reagent to an aldehyde resin [28]. 

To demonstrate the utility of this handle, p-bromobenzoic acid was 

Scheme 5 


Scheme 6 

attached to the support and cleavage was accomplished in TBAF in 

DMF at 65jC or CsF in DMF at 90jC in 78% and 77% yield, respectively 

(Scheme 7). 

Redox-sensitive resin 24 designed for solid-phase peptide synthesis 

(SPPS) [29] was prepared from commercially available 2,5-dimethylbenzoquinone 

in seven steps [30] and loaded to a support via a Wittig 

reaction. Release of the peptide occurs using two sequential mild 

conditions, reduction with NaBH4 followed by TBAF-catalyzed cyclic 

ether formation (Scheme 8) which provide orthogonality to acid sensitive 

reactions. 

Allylic hydroxycrotyl-oligoethylene glyco-n-alkanoyl (HYCRON) 

linker 25 was applied to the synthesis of protected peptides and glycopeptides 

[31]. HYCRON is stable to both acidic and basic conditions and is 

compatible with Boc- and Fmoc-based chemistry. The preparation of this 

novel linker is only two steps from commercially available materials. 

HYCRON linker can be cleaved under neutral conditions using Pd catalyst 

(Scheme 9). 

Scheme 7 


Scheme 8 

III. RESINS AND LINKERS FOR GENERATION 

OF AMIDE FUNCTION 

Functionalized supports with amino groups such as benzhydrylamine 

(BHA) 26 [32] and 4-methylbenzhydrylamine (MBHA) 3 [3] provided Cterminal 

amides upon HF cleavage (Fig. 2). Polyalkoxyaminobenzyl and 

alkoxydiphenylamino resins such as PAL (5-(4-aminomethyl-3,5-dime- 

Scheme 9 


Figure 8 

thoxyphenoxy)valeric acid) 27 [33], Rink amide support (RAM) 28 [34] 

and 4-(4V-methoxyvenzhydryl)phenoxyacetic acid (Dod) linker 29 [35] 

contain more electron-donating groups and were designed on the same 

principles as discussed above for the hydroxymethyl supports (Fig. 8). 

These three linkers are the most widely used in SPOS and require TFA for 

cleavage. Xanthone-based handles XAL (xanthenyl amide linder) 30 [36] 

and Seiber 31 [37] resin were designed to release amides using low 

concentrations of TFA (Fig. 9). Handles which contain an aminomethyl 

and o-nitrobenzyl function (Nb [nitrobenzyl] 32 [38], NBHA [nitrobenzylamine] 

33 [39], and a-methyl-6-nitroveratrylamine) 34 [40] are cleaved by 

photolysis and are based upon the same principles discussed for hydroxyl 

resins (Fig. 10). 

Figure 9 


Figure 10 

In an extension to the xanthenyl theme, the benzyl hydrogen was 

replaced with a substituted p-methoxyphenyl ring to give linker 35 (Scheme 

10) [41]. Peptide amides were cleaved rapidly and in high purity with TFA- 

DCM (1:9) for 15 min or as a protected fragment with TFA-DCM (1:99) 

for 3–10 min. 

Silyl-derived linker 36 was prepared in three steps from a silyl ether of 

serine and incorporated for Fmoc/tBu-based assembly of protected glycopeptide 

blocks (Scheme 11) [42]. The a-carboxylic acid function of serine 

was protected as an allyl ester. Deprotection by a Pd(0) catalyst in the 

presence of dimedone liberated the carboxylic acid in order for subsequent 

Scheme 10 


Scheme 11 

coupling with amines, alcohols, and carbohydrates. The final glycopeptide 

product was released from the support by fluoridolysis (CsF). 

As an extension to the p-carboxybenzenesulfonamide ‘‘safety-catch’’ 

linker [43,44], alkanesulfonamide handle 37 was developed [45]. This linker 

tethers carboxylic acids to the solid support to give an acylated sulfonamide 

which is stable to both basic and acidic conditions (Scheme 12). 

Products were released by treatment with iodoacetonitrile followed by the 

addition of a nucleophile. 

Aryl hydrazide linker 38 stable to both acid and base was utilized in 

SPPS [46]. Treatment of the resin with a copper(II) catalyst in the presence 

of a base and nucleophile gave the corresponding acid, amide, or ester 

(Scheme 13). 

Scheme 12 


Scheme 13 

IV. RESINS AND LINKERS FOR N-SUBSTITUTED 

AMIDE GENERATION 

Linker 39 with an aldehyde attachment point permits amine anchoring 

via reductive amination (Fig. 11) [47]. In peptide synthesis, the handle 

attaches the amino as opposed to carboxylic acid function of the Cterminal 

residue to the support followed by chain elongation (attachment 

to the peptide occurs via a backbone nitrogen). The same strategy for 

developing handles functionalized with an aldehyde is similar to the 

concepts described above. Backbone amide linker (BAL) 40 was prepared 

from the Fmoc-based tris(alkoxy)benzylamide handle PAL [48]. In 

peptide synthesis, BAL allows the preparation of sequences having a 

variety of C-terminal functionalities such as alcohols, N-alkyl amides, 

and head-to-tail cyclic peptides that are devoid of a trifunctional amino 

acid. Due to the electron-donating groups contained in the handle, 

release of the peptide is accomplished with a high concentration of 

TFA. Based upon the BAL concept, the acid sensitive methoxybenzal- 

Figure 11 


Scheme 14 

dehyde polystyrene resin (AMEBA) 41 was reported for the solid-phase 

synthesis of sulfonamides, amides, ureas, and carbamates [49]. Reductive 

amination of aldehydes and ketones with sodium cyanoborohydride 

to Rink amide linker generated N-alkyl amines. Acylation followed by 

cleavage with TFA provided a method to generate a series of difunctional 

amines and N-substituted amide derivatives [50]. Subsequently, 

backbone linker 42 for Boc-based peptide was developed from a 4alkoxybenzyl 

derivative in which products were released upon HF 

treatment (Scheme 14) [51]. 

Contrary to an alkoxy benzene scaffold, secondary amides were 

generated via novel aldehyde linker 43 based upon an indole scaffold 

(Scheme 15) [52]. The indole resin was prepared from indole-3-carboxyaldehyde 

in two steps and reacted with amines under reductive conditions 

to generate resin-bound secondary amines. Treatment of the resin with 

Scheme 15 


Scheme 16 

various reagents followed by TFA yielded amides as well as sulfonamides, 

carbamates, and ureas in high yields. 

MAMP (Merrifield, Alpha-MethoxyPhenyl) resin 44 is an alternative 

to aldehyde linkers to construct N-substituted amides [53]. Nucleophilic 

displacement of the benzylic chloride with an amine followed by 

acylation yielded a secondary amide which was released upon a low 

(f10%) concentration of TFA (Scheme 16). 

V. RESINS AND LINKERS FOR HYDROXYL 

AND GENERATION OF AMINO FUNCTION 

Dihydropyran (DHP) linker 45 is a common handle that couples an 

alcohol to a solid support with subsequent release upon mild TFA conditions 

(Fig. 12) [54]. An alternative approach is to prepare an active 

carbonate linker. N,NV-Disuccinimidyl carbonate (DSC), a valuable 

reagent for converting hydroxymethyl-based supports to their corresponding 

carbonates, was reacted with 4-hydroxymethylpolystyrene 46 

and 4-nitrobenzamido (Nbb) 47 resins to anchor alcohols and phenols 

(Scheme 17) [55]. The final products were released from the solid support 

by HF and photolysis, respectively. 


Figure 12 

p-Benzyloxybenzylamine (BOBA) 48 is a new class of an amine 

support and was prepared from Merrifield resin in two steps [56]. BOBA 

resin was treated with an aldehyde in the presence of an acid to give an 

imine that subsequently reacted with Yb(OTf )3-catalyzed silyl enolates 

(Scheme 18). Cleavage with trimethylsilyl triflate (TMSOTf) or 2,3dichloro-5,6-dicyano-1,4-benzoquinone 

(DDQ) gave either phenols or 

amines, respectively. 

9-Phenylfluoren-9-yl polystyrene (Phfl) based resin 49 was applied in 

the solid-phase synthesis of hydroxyl and amino functions [57,58]. This 

resin has higher acid stability compared to the structurally similar trityl 

resin. Final release of the product is accomplished with TFA in high purity 

(Scheme 19). 

Trialkylsilane resin (PS-DES) 50 was incorporated for solid-phase 

glycosylation by anchoring a glycosyl donor via their corresponding thiophenyl 

ether or h-glucopyranosyl fluorides (Scheme 20) [59]. Disaccharides 

Scheme 17 


Scheme 18 

were prepared by reaction with a glycosyl acceptor followed by cleavage 

with acetic acid (AcOH). 

Phenols were constructed form novel serine-derived handle 51, 

which was stable to acids (TFA) and bases (pyridine) (Scheme 21) [60]. 

The final products were released from the support by fluoride ion. 

A variety of cleavage conditions have been reported for the release 

of amines from a solid support. Triazene linker 52 prepared from 

Merrifield resin in three steps was used for the solid-phase synthesis of 

aliphatic amines (Scheme 22) [61]. The triazenes were stable to basic 

conditions and the amino products were released in high yields upon 

treatment with mild acids. Alternatively, base labile linker 53 synthesized 

from a-bromo-p-toluic acid in two steps was used to anchor amino 

functions (Scheme 23) [62]. Cleavage was accomplished by oxidation of 

the thioether to the sulfone with m-chloroperbenzoic acid followed by helimination 

with a 10% solution of NH 4OH in 2,2,2-trifluoroethanol. A 

linker based on 1-(4,4V-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) 

primary amine protecting group was developed for attaching amino 

functions (Scheme 24) [65]. Linker 54 was stable to both acidic and 

basic conditions and the final products were cleaved from the resin by 

treatment with hydrazine or transamination with n-propylamine. 

Scheme 19 


Scheme 20 

REM linker 55 (regenerated after cleavage, functionalized by 

Michael addition) is a traceless handle for anchoring secondary amines 

(Fig. 13) [64]. Tertiary amines were prepared on this linker via basedinduced 

Hofmann elimination of the subsequent quaternary ammonium 

salt. An analogous vinyl sulfone linker was prepared from Merrifield resin 

to perform the identical synthetic strategy (Scheme 25) [65]. Similar to 

REM, vinyl sulfone handle 56 was regenerated following cleavage, but 

was more stable to acids and nucleophiles such as Grignard reagents than 

the former. An extension to the vinyl sulfone theme was demonstrated by 

inserting a carbamate function at the anchoring position for the assembly 

of 2- and 2,4-substituted pyrrolidines (Scheme 26) [66]. The acid stable, 

base labile (final cleavage accomplished with NaOMe) support 57 was 

used for N-acyliminium ion reactions. 

Scheme 21 


Scheme 22 

Scheme 23 

Scheme 24 


Figure 13 

Scheme 25 

Scheme 26 


Scheme 27 

N-Protected amines were assembled on solid-phase via sulfonamidebased 

handle 58 (Scheme 27) [67]. Tertiary sulfonamides were generated 

upon reaction with allylic, benzylic and primary alcohols under Mitsunobu 

conditions. Secondary amines were released from the support using 

mild nucleophilic conditions such as treatment with thiophenol and 

potassium carbonate. 

A versatile approach for the solid-phase synthesis of aminopyridazines 

used the anchoring of 3,6-dichloropyridazine to resin-bound 

thiophenol 59 (Scheme 28) [68]. Treatment with nucleophilic amines 

released the aminopyridazine products from the solid support without 

further oxidation. 

Traceless linker 60 based on a benzotriazole scaffold was reacted with 

amines and aldehydes to produce Mannich-type amine products [69]. Final 

product release was achieved by treatment with Grignard reagents 

(Scheme 29). 

Scheme 28 

Scheme 29 


Figure 14 

VI. RESINS AND LINKERS FOR HYDROXAMIC 

GENERATION OF ACID FUNCTIONS 

Hydroxamic acids are an important class of compounds targeted as potential 

therapeutic agents. N-Fmoc-aminooxy-2-chlorotrityl polystyrene 

resin 61 allowed the synthesis and subsequent cleavage under mild conditions 

of both peptidyl and small molecule hydroxamic acids (Fig. 14) 

[70]. An alternative hydroxylamine linkage 62 was prepared from trityl 

chloride resin and N-hydroxyphthalimide followed by treatment with 

hydrazine at room temperature (Scheme 30) [71]. A series of hydroxamic 

acids were prepared by the addition of substituted succinic anhydrides to 

the resin followed by coupling with a variety of amines, and cleavage with 

HCOOH-THF(1:3). 

Scheme 30 


VII. RESINS AND LINKERS FOR GENERATION 

OF SULFONAMIDE, UREA, AND 

GUANIDINE FUNCTIONS 

Aminosulfonyl ureas were constructed from a sulfonylcarbamate linkage 

(Scheme 31) [72]. Reaction of chlorosulfonyl isocyanate (CSI) with 

Wang resin provided a chlorosulfonylcarbamate 63 which was then 

converted to substituted amino sulfonylcarbamate compounds by reaction 

with excess amines. The final aminosulfonyl urea products were 

cleaved from the resin by treatment with amines in HF at reflux 

temperature for overnight. 

Urea libraries were assembled via thiophenoxy carbonyl linker 64 

readily available in two steps from Merrifield resin (Scheme 32) [73]. 

Treatment of this linker with primary or secondary amines, followed by 

basic cleavage with amines generated the ureas. An alternative 

approach for the synthesis of ureas was to treat p-nitrobenzophenone 

oxime resin with phosgene to give p-nitrophenyl(polystyrene)ketoxime 

(Phoxime resin) 65 [74]. The addition of primary amines to the 

phosgenated oxime linker gave a resin-bound carbamate. Ureas were 

genated by reaction with a second set of amines at temperatures greater 

than 80jC (Scheme 33). 

Traditional SPPS anchors the peptide to the support via the acarboxylic 

acid of the C-terminal residue. Novel sulfonyl linker 66 was 

prepared to side-chain anchor the guanidine function of arginine 

(Scheme 34) [75]. To demonstrate the utility of the linker, tripeptide 

H-Phe-Arg-Ala-OMe was assembled in which amino acids were 

extended to the anchoring residue in both the C- and N-terminal 

directions. HF cleavage released the peptide from the support. Small 

molecules containing guanidines were constructed from carbonylimidazole 

handle 67 generated from Wang resin (Scheme 35) [76]. Treatment 

of the carbonylimidazole linker with thiourea basic conditions afforded 

Scheme 31 


Scheme 32 

Scheme 33 

Scheme 34 


Scheme 35 

a resin-linked thiourea product. Sulfur displacement of the thiourea 

resin with primary and secondary amines followed by TFA cleavage 

provided guanidine-containing products. Both mono- and disubstituted 

guanidines were prepared in good yields and purities using acyl isothiocyanate 

resin 68 prepared from carboxypolystyrene in two simple steps 

(Scheme 36) [77]. Reaction of a variety of amines with this resin produced 

the corresponding acyl thioureas under mild conditions. The guanidine 

moiety formation was achieved by exposing the acyl thiourea resin to a 

primary or secondary amine. Cleavage of the acyl guanidine was effected 

by treatment with TFA-CHCl3-MeOH (1:1:1). 

VIII. RESINS AND LINKERS FOR GENERATION 

OF ALDEHYDE FUNCTIONS 

The Leznoff acetal linker 69 was used to anchor an aldehyde to the solid 

support and following a series of reactions, the aldehyde was released by 

acidic cleavage [78]. An application of this resin was demonstrated for a 

biaryl aldehyde library synthesis which incorporated a Suzuki–Miyaura 

reaction (Scheme 37) [79]. Cleavage was effected by a solution of 3 M HCl 

Scheme 36 


Scheme 37 

at 80jC to give excellent yields for most of the products. An alternative 

strategy implemented serine or threonine as a linker to anchor an aldehyde 

to the solid support [80]. Unlike the acetal formation described above, this 

linker reacts with an aldehyde to form an oxazoline with release from the 

support by aqueous acid (such as HOAc) at 60jC. 

A second strategy employed tartaric acid-based linker 70 prepared 

from an amino PEGA resin in which C-terminal a-oxo-aldehydes were 

generated by an oxidative cleavage (Scheme 38) [81]. Following linear 

assembly of the peptide by Fmoc chemistry, TFA treatment removed the 

side-chain protecting groups and converted the anchoring acetonide to a 

1,2-diol which was oxidized to the aldehyde with NaIO4. 

IX. RESINS AND LINKERS FOR GENERATION 

OF OTHER FUNCTIONS 

Cleavage of all the linkers described above provide a functional group 

(carboxylic acid, amide, amine, etc) at the anchoring position. Silyl-based 

handles 71,72, and 73 as well as germanium-based handle 74 insert a C-H 

bond at the anchoring position and are referred to as traceless (Fig. 15) [82– 

Scheme 38 


Figure 15 

85]. A further extension to this concept was (dimethylsilyl)propionic acid 

linker 75 used for the solid-phase synthesis of aryl-containing organic 

compounds [86]. The linker was cleaved smoothly with TFA and has been 

used for the synthesis of compounds which involved alkylation, acylation, 

and Mitsunobu reactions. 

Silicon linker 76 was used for direct loading of aromatic compounds 

to supports for the assembly of pyridine-based tricyclics (Scheme 

39) [87]. Following the initial coupling of an aromatic bromide to the 

resin by halogen/metal exchange in the presence of tert-butyl lithium, a 


Scheme 39 

series of reactions including TFA deprotection of Boc, alkylation under 

strong basic condition, SnCl2 reduction, and ring cyclization were 

performed. The final tricyclic products were released from the polymer 

via basic fluoride (Bu4N + -F) in THF at room temperature. A similar 

trialkylsilane linker was synthesized from Merrifield resin in two steps 

[88]. 

Piperazine linker 77 was treated with propargyltriphenylphosphine 

bromide to provide a resin-bound Wittig reagent (Scheme 40) [89]. Base 

treatment followed by aldehyde addition produced a resin-bound 2-aminobutadiene 

which was implemented in [4+2] cycloadditions. Alternatively, 

treatment with 3% TFA in CH2Cl2 released a,h-unsaturated methylketones 

in high yields. 

Scheme 40 


Scheme 41 

Acetal handle 78 synthesized from Merrifield resin and 4-hydroxybenzaldehyde 

was applied to the solid-phase synthesis of carbohydrates 

and 1-oxacephams (Scheme 41) [90]. For the latter, a 1,3-diol was 

initially anchored to the support to form a cyclic acetal. A ring opening 

reaction with DIBAL generated a resin-bound alcohol which was 

converted to the corresponding triflate for N-alkylation with 4-vinyloxyazetidin-2-one. 

A Lewis acid catalyzed ring closure released 1-oxacephams 

from the support. 

Aryl hydrazide-based linker 79 was developed as a traceless handle 

that released products under mild oxidative conditions (Scheme 42) [91]. 

Polymeric bound p–iodophenylhydrazide was subjected to a variety of 

Pd 0 -catalyzed coupling reactions (Heck, Suzuki, Sonogashira, and Stille). 

Oxidation with Cu(OAc)2 in MeOH and pyridine released the final 

products in 50–96% yield. 

A traceless linker for solid-phase homo- and hetero-Diels-Alder 

reactions was based upon resin bound quinodimethane precursors 

Scheme 42 


Scheme 43 

(Scheme 43) [92]. Reaction of dienophiles such as 4-nitrobenzaldehyde 

with linker 80 at high temperature gave Diels-Alder products. Dihydropyrans 

were released from the support by Bronsted or Lewis acid-nucleophile 

combinations in moderate to good yield with stereoselectivity for the 

anti isomer. 

X. CONCLUSION 

The past decade witnessed a renaissance in drug discovery due to the 

emergence of solid-phase synthesis. Initially, the solid supports and linkers 

used for the repetitive process of biomolecule assembly applied to the 

construction of small molecule libraries and scaffolds were required to 

contain a carboxylic acid or amide in order to anchor to the polymeric 

support. Thus, the linkers from solid-phase peptide synthesis such as Rink, 

Wang, and PAL were commonly employed in the synthetic strategy. As 

new bond-forming reactions have been adapted for solid phase as well as 

the construction of novel lead compounds, synthetic pathways are requiring 

additional handles that release compounds into solution upon various 

cleavage conditions and provide additional functionality at the anchoring 

position. In a retrosynthetic analysis of a library, one should plan the 

anchoring strategy as it relates to the functionality of the molecule as well 

as to insure that the cleavage conditions are compatible with the synthetic 

scheme. Although there are now a plethora of linkers that have been 

described in the literature, novel handles still provide medicinal chemists 

the tools to expand molecular diversity with the ultimate reward of 

discovering a new drug candidate. 


Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved. 

Appendix A. HANDLES AND DERIVATIZED 

SOLID SUPPORTS







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8 Allosteric Modulation 

of G-Protein-Coupled 

Receptors: Implications 

for Drug Action 

Angeliki P. Kourounakis 

University of Thessaloniki, Thessaloniki, Greece 

Pieter van der Klein and Ad P. IJzerman 

Leiden University, Leiden, The Netherlands 


Representing one of the largest superfamilies of proteins in the human 

body, G-protein-coupled receptors (GPCRs) play a crucial role in the regulation 

of a variety of physiological processes, particularly within the 

central nervous system and the cardiovascular and endocrine systems. It is 

estimated that this superfamily comprises about 500 and possibly over 1000 

receptor (sub)types having similar structural and/or sequence motifs, while 

operating via common transduction mechanisms to mediate the transmission 

of extracellular signals into biochemical or electrophysiological 

responses in a cell. A specific endogenous molecule, such as a neurotransmitter 

or hormone, acts as the signaling species that binds to the receptor, 

resulting in an activation of intracellular G proteins and signal propagation. 

Hence, GPCRs are important drug targets; approximately 60% of 

current drugs produce their therapeutic actions by binding to GPCRs. 


The design and development of synthetic drugs is mainly focused on 

mimicking (as in the case of agonists) or blocking (as in the case of 

antagonists and inverse agonists) the action of the endogenous signaling 

molecule by competing at the same site (the ligand binding site) ona 

specific receptor. Recently, a new concept of interference with drug action 

at GPCRs has emerged for some receptor subclasses such as the muscarinic 

or adenosine receptor. This concept, namely allosteric modulation of 

the receptor by molecules binding at a second (allosteric) site, is thus far 

relatively unexplored for GPCRs, although relatively common in the 

family of ion channel receptors [1]. This indirect (allosteric) mechanism 

(i.e., the modulation of the efficacy or affinity of the endogenous ligand for 

its receptor) is the molecular basis of the therapeutic action of benzodiazepines 

that interact with g-aminobutyric acid A (GABAA) receptors 

coupled through ion channels. In contrast, there has been no therapeutic 

role found for directly acting agonists or antagonists on this receptor. 

Nevertheless, only a few drugs, such as gallamine, alcuronium, or 

pancuronium, are known to exert their action at an allosteric site on a 

GPCR [2]. 

The potential advantages or benefits of allosteric drugs over agonists, 

antagonists, and inverse agonists may be elaborated as follows. 

It is generally found that within the GPCR family, subtypes exist 

that bind the same signaling molecule but have different tissue distributions 

as well as functions. These receptor subtypes have often a high 

sequence homology, especially in the regions of the receptor that are 

thought to contain the ligand binding site. Thus, in most cases it has been 

proven difficult to develop drugs that not only are highly selective for one 

receptor subtype but have highly controlled effects on the function of that 

receptor and act in those tissues only where their action is desired. 

An allosteric drug has the following properties: 

1. Has no action when binding on its own to the receptor but only 

modulates the actions of the naturally occurring hormone or 

neurotransmitter when it is released. Therefore, the temporal 

aspects of the natural signaling mechanism are retained and 

desensitization is minimized. 

2. Has a defined maximum effect that is determined by the 

cooperativity associated with its allosterism. 

3. Can act selectively at various receptor subtypes not only by 

means of its own affinity but also on cooperativity. No pharmacological 

agent has yet exploited the latter property. 


4. Has enhancing properties that can selectively intensify a 

weakened signal from a specific receptor subtype, alleviating the 

effects caused by a localized neurotransmitter deficit such as in 

Alzheimer’s or Parkinson’s disease. 

Although allosteric sites have been characterized on certain biogenic 

amine receptors—muscarinic acetylcholine, dopamine, and a-adrenergic 

receptors—as well as on adenosine receptors, it is not yet known whether 

the presence of an allosteric site is a characteristic of only a few, all, or 

subsets of GPCRs. Furthermore, a relevant question is whether these sites 

might have a physiological regulatory role as a consequence of binding 

with endogenous molecules. Interestingly, there are a few recent reports 

showing endogenous ligands affecting GPCR binding and function allosterically. 

The endogenous tetrapeptide Leu-Ser-Ala-Leu, released from 

nerve terminals upon depolarization, inhibits 5HT1B receptor binding and 

function at nanomolar concentrations [3,4], an effect specific at 5HT1B and 

not at other 5-hydroxytryptamine receptors that were examined. Also, a 

recent study reported that binding and function of the human oxytocin 

receptor can be inhibited directly by nanomolar concentrations of 5hdihydroprogesterone 

[5]. Although the actions of the peptide and the 

steroid have a number of common features that make their interactions 

different from those previously observed, both studies suggest an entirely 

unanticipated cross talk between very different signaling mechanisms, the 

consequences of which are not yet known. 

II. DEFINITION OF ‘‘ALLOSTERIC’’: 

RELATED MODELS 

The term ‘‘allosteric’’ was first introduced by Monod and Jacob [6], who 

referred to an allosteric inhibition (of the synthesis of a tryptophan 

precursor by tryptophan) in describing the mechanism underlying the 

action of ‘‘an inhibitor that was not a steric analog of the substrate.’’ 

Thus, first introduced in the field of enzymology, the term ‘‘allosteric’’ 

(Greek aEEo, other, different; jH eUeo, solid, shape) means ‘‘having a 

different shape.’’ It soon referred to the presence in an enzyme of a 

(secondary) site of attachment for a substance that modifies enzyme 

activity without interacting directly with the active center (primary site). 

The allosteric effect, therefore, was attributed to a change in either the 

three-dimensional structure of the peptide chain or else a change in 


conformation (allosteric transition) that affects the binding of the substrate 

to the active site [7]. 

Thus, the emphasis was shifted toward the crucial role of conformational 

changes of proteins, as elaborated by Monod et al. in 1965 [8]. The 

concept extended from enzymology to ‘‘receptology,’’ first to ion channel 

receptors and subsequently to GPCRs. The primary site on a receptor is 

thus referred to as the (classical) ligand binding site or ‘‘orthosteric site,’’ 

while the secondary site, or allosteric site, affects binding at the primary 

site by inducing a conformational change in the tertiary structure of the 

receptor protein. 

The simplest model that can describe allosteric interactions at 

GPCRs is the ternary complex allosteric model [9]. As shown in 

Figure 1, according to this model two parameters define the actions of 

allosteric agent (X): its affinity for the unoccupied receptor (Kx) and its 

cooperativity (a) with the ligand (A) that interacts at the primary binding 

site: a1, 

positive cooperativity. 

However, based on the concept that GPCRs are able to adopt a 

variety of conformations, an extended model can also be described, as 

shown in Figure 2. In this extended ‘‘cubic ternary complex model’’ of 

receptor activation and modulation, the receptor can interconvert between 

an active (R * ) and an inactive conformation (R), each with a different 

Figure 1 Representation of the simple ternary allosteric complex model of 

interaction of a ligand A with an allosteric agent X at a receptor R. (From Ref. 2.) 


Figure 2 Representation of a ‘‘cubic ternary complex’’ model of allosteric 

interaction: R, the inactive state of the receptor; R*, the active state of the 

receptor; A, ligand; X, allosteric agent. (From Ref. 14.) 

affinity for the G protein, ligand A, and allosteric modulator (X). Relative 

stoichiometry of the states would be determined by the presence of G 

protein and agonists and modified by allosteric modulators [10]. 

III. MUSCARINIC AND ADENOSINE RECEPTORS 

Allosteric interactions on GPCRs have been observed for the muscarinic 

[11–13], adenosine A 1 [14], a 2A-adrenergic [15–17], and dopamine D 2 

receptor [18]. This chapter focuses only on two allosteric phenomena, as 

well as their potential for therapeutic exploitation: that on the muscarinic 

receptor and that on the adenosine receptor. 

A. Allosteric Modulation on the Muscarinic Receptor 

The first and best-studied allosteric site on GPCRs is that on the muscarinic 

receptor [9,10,12,19,20]. For the five subtypes of these receptors that 

have been cloned and pharmacologically defined as M1 to M5, various 

agents have been identified that allosterically regulate selectively these 


eceptor subtypes [19,21]. Gallamine was the first compound shown to 

interact allosterically with the muscarinic (M2) receptor, exhibiting negative 

cooperativity with antagonists [ 3 H]NMS and [ 3 H]QNB [22,23]. The 

interactions of gallamine with the M2 receptor were shown to agree with 

the ternary complex allosteric model in both binding and functional studies 

[23–25]. Since then, a number of ligands have been discovered that interact 

with various muscarinic receptor subtypes, confirming that the allosteric 

site is present on all five subtypes. 

Furthermore, these allosteric effects were shown to be truly subtype 

specific, depending on the nature of the allosteric modulating compound. 

Thus, alcuronium exerts positive copperativity with [ 3 H]NMS at the M2 

and M4 but not at the M1 and M3 receptors [26,27], while other neuromuscular 

junction blockers such as stercuronium, pancuronium, and dtubocurarine 

have been shown to exhibit their effects via an allosteric 

mechanism specifically on the M 2 receptors [28–30]. 

A growing number of other diverse compounds have also been 

shown to bind to an allosteric site on the muscarinic receptors. Among 

them are pirenzepine (highly selective for M1 receptor), lidocaine and 

verapamil (ion channel blockers), tacrine (anticholinesterase compound), 

batrachotoxin, and strychnine (glycine receptor antagonist) 

[25,31–35]. 

Although in the cases of gallamine and some of the other agents, a 

values for various ligands were all below 1, another group of compounds, 

such as brucine and analogues, appear to be allosteric agents exhibiting 

positive cooperativity at one or more muscarinic receptor subtypes [36,37]. 

The interest in agents positively cooperative with ACh at muscarinic 

receptors stems from their potential use in the treatment of cognitive 

deficits such as Alzheimer’s disease. Brucine and analogues were shown not 

only to enhance the affinity of ACh in radioligand binding studies for the 

M1,M3, and M4 muscarinic receptors but further to modulate the actions 

of acetylcholine in functional studies. First in GTPase and [ 35 S]GTPgS 

binding assays (Fig. 3), then in cAMP production and intracellular 

Ca 2+ mobilization assays (Fig. 4), and finally in a tissue model of contraction 

of the guinea pig ileum strip (Fig. 5) [9]. In all cases, the activity 

of these analogues showed ‘‘absolute subtype selectivity’’ with variable 

effects on the various muscarinic subtypes. The results suggested the pharmacological 

feasibility of selectively elevating subnormally functioning 

cholinergic neurons in the central nervous system (CNS) by means of an 

appropriate allosteric enhancer. 


Figure 3 Enhancement by brucine of ACh potency at M1 receptors in functional 

assays in membranes. Brucine (100 AM) increased the potency of ACh to 

stimulate [ 35 S]GTPgS binding to G proteins in m 1 CHO cell membranes. In this 

experiment, the EC50 value for ACh decreased from 2.8 AM (5) to0.9AM (n) 

without significantly affecting the basal response or maximal stimulation. (From 

Ref. 9.) 

B. Allosteric Modulation on the Adenosine Receptor 

Extracellular adenosine is regarded as a local hormone that exerts numerous 

physiological actions in a variety of mammalian tissues. The actions of 

this nucleoside in the body are mediated by G-protein-coupled adenosine 

receptors subclassified as A 1,A 2A, A 2B, and A 3 [38]. The adenosine A 1 

receptor is interfaced with a Gi protein, which is negatively coupled to the 

adenylate cyclase–cAMP signal transduction pathway, and thus, upon 

activation, leads to a reduction in intracellular cAMP levels. This receptor 

is highly and widely expressed in not only in the CNS but also in other 

tissues such as fat cells, bladder, and heart [38–40]. A variety of adenosinemediated 

efffects (hypotension, inhibition of lipolysis, analgesia) occurs via 

the adenosine A 1 receptor, rendering it an important target for pharmacological 

intervention. Nonetheless, the wide distribution of adenosine 


Figure 4 Dose–response curves for the potentiation by brucine of ACh wholecell 

M1 muscarinic receptor responses. (A) Brucine (10 4 M) enhanced the 

potency of ACh to increase cAMP accumulation in M1 CHO cells by 2.6-fold. (B) 

Brucine (100 AM) produced a 3.0-fold increase in ACh potency in Ca 2+ response 

to ACh. (From Ref. 9.) 


Figure 5 (A) In a dose-dependent manner, N-chloromethyl brucine (CMB) 

enhanced the field-stimulated contractions of isolated guinea pig ileum strips. 

The contractions were inhibited by atropine (30 nM). (B) Histogram of the 

percentage enhancement of contraction produced in four independent experiments 

of the type illustrated in A. (From Ref. 9.) 


eceptors offers both opportunities and drawbacks for therapeutic intervention 

[38,41]. For example, A1 adenosine agonists, through their interaction 

with adenosine A1 receptors on fat cells, are able to reduce free fatty 

acid levels in the blood. Since this effect sensitizes insulin’s action [42]. It 

may be a very useful feature in non-insulin-dependent diabetes mellitus 

(type II diabetes). However, serious side effects occur by the concomitant 

bradycardia and drop in mean arterial pressure due to interference with 

cardiovascular adenosine receptors [43]. Various strategies have been followed 

to circumvent all or some of these problems, such as the development 

of partial agonists for that purpose [44–48]. It was shown that some 

of these compounds were virtually ‘‘silent’’ on the heart, while keeping a 

pronounced, full effect on adipose tissue [49]. 

On the other hand, among the effects of receptor-bound adenosine 

is the ability to protect organs, including the heart and brain, from 

ischemic injury [50–52]. The formation of extracellular adenosine as a 

breakdown product of ATP is a local phenomenon, induced by a tissue 

at risk (e.g., under hypoxic or anoxic conditions: heart failure, stroke, 

etc.). As a consequence, compounds that would increase adenosine’s 

concentration, and thus its tissue-protective effect, might have a better 

therapeutic profile than the agonists described earlier. Marketed nucleoside 

transport blockers such as dipyridamole and dilazep have already 

proven this concept by inhibiting the intracellular uptake of extracellular 

adenosine, and thereby effectively increasing its concentration outside 

the cell [53,54]. 

Another interesting approach is to enhance adenosine’s action 

locally by means of an allosteric enhancer. In 1990, Bruns and coworkers 

reported on various 2-amino-3-benzoylthiophene derivatives 

capable of enhancing the binding and activity of reference A1 receptor 

agonists, such as N 6 -cyclopentyladenosine (CPA) [14,55]. One of these 

‘‘allosteric modulators,’’ PD81,723, or (2-amino-4,5-dimethyl-trienyl) 

[3-(trifluoromethyl) phenyl]methanone (Fig. 6), has been investigated 

pharmacologically in greater detail by various independent research 

groups [56–61]. 

The modulator PD81,723 enhances two- to threefold the binding 

and function of agonists such as CPA, R-PIA, or NECA to adenosine 

A1 receptors [62]. As shown in Figure 7, in displacement experiments of 

[ 3 H]DPCPX from the human adenosine A1 receptor (wild type), the binding 

curve of CPA in the presence of PD81,723 is shifted leftward; it seems 

that CPA binds more efficiently, since lower concentrations of this agonist 

are needed to displace the same concentration of radioligand. This 


Figure 6 Structure of PD81,723, (2-amino-4,5-dimethyl-trienyl)[3-(trifluoromethyl) 

phenyl]methanone and adenosine A1 agonists/antagonists. 

Figure 7 Displacement of 0.4 nM [ 3 H]DPCPX by various concentrations of CPA 

from human wild-type (CHO A 1) and mutant (CHO A 1-mutT277A) adenosine A 1 

receptors in the absence (n) or presence (5) of PD81,723 (10 AM). 


‘‘enhanced’’ activity of CPA is also maintained in second messenger 

assays, where, for example, lower concentrations of CPA (in the presence 

of PD81,723) are needed for the inhibition of forskolin-stimulated 

cAMP production in cells bearing adenosine A1 receptors (Fig. 8). It is 

known that PD81,723 slows down the kinetics (dissociation) of 3 H-labeled 

agonists such as [ 3 H]CHA or [ 3 H]CCPA from the receptor as shown in 

Figure 9; the half-life of 17 min for the dissociation of CCPA alone from 

the rat A 1 receptor is increased to 25 min in the presence of 10 AM 

PD81,723 [63]. It is postulated that this compound binds to an allosteric 

site on the adenosine A1 receptor—which, unlike the muscarinic one, is 

not yet so well defined—while at somewhat higher concentration it binds 

to the ligand binding site exhibiting antagonistic action. It is presumed 

that via its allosteric activity PD81,723 increases the proportion of adenosine 

receptors in the ‘‘active’’ (R * ) conformation that has a high affinity 

for agonists and low for antagonists and inverse agonists (Fig. 2). Not 

only are these effects selective for the A 1 receptor, but they disappear 

upon a mutation of the receptor at the proposed agonist binding site [62]. 

Threonine at position 277 on the A1 receptor is considered to interact with 

ribose ring of agonists, since changing it to alanine greatly decreases the 

affinity for agonists but not for antagonists. This mutation also eliminated 

the activity of PD81,723 (Fig. 7), which no longer can increase the already 

Figure 8 Forskolin-stimulated cAMP production of CHO A 1 cells after addition 

of CPA in the absence (n) or presence (5) of10AM PD81,723. 


Figure 9 Dissociation of agonist [ 3 H]CCPA from rat brain A1 receptors in the 

presence (5) or absence (n) of10AM PD81,723. 

low affinity of agonists [64–67]. This indicates that an intact agonist binding 

site of the receptor is required for PD81,723 to exert its allosteric 

action [62]. 

Recently we developed a series of novel PD81,723 analogues, 

some of which appear to be superior to PD81,723 in their enhancing 

activity [68,69]. The synthesis of these derivatives is relatively straightforward, 

as shown in Figure 10 [68–72]. The 4,5-dimethyl group and 

the benzoyl moiety were targets for further modifications, leading to 

series of 4,5-dialkyl (1a–g), of tetrahydrobenzo (1h–u) and of tetrahydropyridine 

(3a–g) derivatives (Fig. 10, Tables 1 and 2). These derivatives 

were evaluated both as allosteric enhancers of agonist binding to 

the rat adenosine A1 receptor and as antagonists on this receptor. 

Among them, a number of compounds, in particular 1b, 2e, 1j, 1n, 

and 1u (Fig. 11, Table 1), proved to be superior to the reference 

compound (PD81,723) in both enhancing activity and diminished antagonistic 

behavior [68]. 

Some structure–activity relationships of a further developed R 4 , R 5 

alkyl/cycloalkyl series (2a–o, Fig. 10, Table 1) were also investigated. 

This study [69] revealed structural features that favored allosteric 

enhancing activity, such as benzoyl lipophilic substitution and thiophene 

4-alkyl substitution, while other features, such as thiophene 5-bulky 

substitution, favored antagonistic properties. Upon further analysis, a 


Figure 10 Scheme of synthesis of PD81,723 analogues. Reagents and 

conditions: i, DMF; S 8,Et 3N, RT (or EtOH, S 8,Et 2NH, 50jC); ii, C 6H 6, h-alanine, 

HOAc; iii, EtOH, S8, Et2NH; iv, BzCl, CH2Cl2, Et3N. 

Table 1 Structure and Enhancing/Antagonistic Activity of 2-Amino-3-benzoylthiophenes 

Analogues 1a–u and 2a–o 

Compound 

R 0 

R 4 

R 5 

Enhancement (%) a 

Antagonism (%) b 

PD81,723 3-CF3 CH3 CH3 100 39 (F4) 

1a H CH3 CH3 8(F5) 14 (F3) 

1b 3-Cl CH3 CH3 80 (F19) 19 (F4) 

1c 4-Cl CH 3 CH 3 93 (F32) 41 (F6) 


Table 1 Continued 

Compound 

R 0 

R 4 

R 5 



PD81,723 3-CF3 CH3 CH3 100 39 (F4) 

1d H CH 2CH 3 CH 3 31 (F4) 13 (F3) 

1e 3-CF3 CH2CH3 CH3 112 (F10) 5 (F11) 

1f 3-Cl CH2CH3 CH3 30 (F7) 22 (F2) 

1g 4-Cl CH 2CH 3 CH 3 97 (F25) 20 (F12) 

1h H —(CH 2) 4— 47 (F4) 35 (F6) 

1i 2-Cl —(CH2)4— 73 (F19) 35 (F3) 

1j 3-CF3 —(CH2)4— 122 (F19) 32 (F8) 

1k 3-Cl —(CH 2) 4— 93 (F6) 51 (F5) 

1l 3-I —(CH2)4— 113 (F18) 66 (F1) 

1m 4-CF3 —(CH2)4— 131 (F11) 57 (F4) 

1n 4-Cl —(CH2)4— 123 (F15) 40 (F5) 

1o 4-Br —(CH 2) 4— 128 (F18) 42 (F4) 

1p 4-I —(CH2)4— 155 (F21) 64 (F8) 

1q 4-NO2 —(CH2)4— 34 (F22) 19 (F2) 

1r 4-CH 3 —(CH 2) 4— 137 (F21) 30 (F3) 

1s 4-CO 2CH 3 —(CH 2) 4— 44 (F9) 29 (n=1) 

1t 4-CO2H —(CH2)4— 29 (F3) nd 

1u 3,4-Cl —(CH2)4— 151 (F24) 35 (F4) 

2a 3-CF 3 H CH 3CH 2CH 2 88 (F8) 52 (F3) 

2b 3-Cl H CH 3CH 2CH 2 67 (F18) 54 (F5) 

2c H H CH3CH2CH2 0(F30) 50 (F7) 

2d 3-CF3 H C5H9 99 (F25) 49 (F2) 

2e 3-Cl H C 5H 9 52 (F12) 64 (F1) 

2f 3-Cl H C6H11 57 (F2) 64 (F3) 

2g H H C6H5 21 (F5) 75 (F2) 

2h 3-Cl H C 6H 5 38 (F6) 80 (F1) 

2i 3-CF 3 H C 6H 5 42 (F7) 58 (F3) 

2j H H (CH3)2CHCH2 7(F14) 47 (F5) 

2k H CH3 CH3CH2 13 (F17) 27 (F7) 

2l H CH 3CH 2CH 2 CH 3CH 2 69 (F19) 17 (F6) 

2m 3,4-Cl CH 3 CH 3 116 (F7) 50 (F1) 

2n 4-tBu CH3 CH3 125 (F24) 47 (F2) 

2o 4-tBu —(CH2)4— 137 (F10) 40 (F4) 

a Enhancing activity (at 10 AM of test compound) is expressed as percentage of decrease (FSEM) in 

[ 3 H]CCPA dissociation over control (0%) and that of PD81,723 (100%, n = 3). 

b Antagonistic activity is expressed as percentage of displacement (FSEM) of 0.4 nM of [ 3 H]DPCPX by 10 

AM of test compound. nd: not determined. 


Table 2 Structure and Enhancing/Antagonistic Activity of 2-Amino-3-benzoyl- 

4,5,6,7-tetrahydrothieno [2,3-c]pyridines 3a–g and 4 

Compound R 0 

R 1 



3a H H 53 (F37) 67 (F5) 

3b H 3-Cl 106 (F27) 80 (F1) 

3c H 4-Cl 69 (F23) 52 (F2) 

3d H 3,4-Cl 57 (F36) 4 (F2) 

3e 4-Cl H 132 (F21) 60 (F0) 

3f 4-Cl 3,4-Cl 106 (F31) 46 (F2) 

3g 3,4-Cl H 174 (F37) 51 (F0) 

4 — — 14 (F27) 72 (F2) 

Theophylline 15 (F7) 56 (F5) 

a Enhancing activity is expressed as percentage of decrease (FSEM) in [ 3 H]CCPA 

dissociation over control (0%) and that of PD81,723 (100%, n = 3). 

b Antagonistic activity is expressed as percentage of displacement (FSEM) of 0.4 nM of 

[ 3 H]DPCPX by 10 AM of test compound. 

significant correlation was found between antagonistic activity and hydrophobic 

fragment constants (k values) [73] of substituent R 5 (Fig. 12), 

in contrast to a negative correlation with those of R 4 . Finally, comparison 

of low energy conformations (Fig. 13) of some of the 2-amino-3benzoylthiophene 

derivatives (PD81,723 and 2f) with known adenosine 

A1 receptor antagonists (theophylline and 8-cyclohexyltheophylline) 

indicated that thiophene 5-substituents (R 5 ) may interact with the same 

lipophilic domain of the adenosine A1 receptor that accommodates 8substituents 

of xanthine antagonists. The separation of the two activities, 

antagonism and allosteric enhancement, is ultimately necessary for 

the development of more potent and selective allosteric enhancers for the 

adenosine A1 receptor with potential therapeutic applications. 


Figure 11 Concentration–effect curves for derivative 1u and PD81,723. 

Enhancement of 100% is expressing the maximum decrease in [ 3 H]CCPA 

dissociation by the highest concentration of 1u. 

Figure 12 Correlation of lipophilicity parameter (k) for substituent R 5 of compounds 

1a,d–f,h, j, k, and 2a–o with their antagonistic activity. ***p < 0.0001. 


Figure 13 Structure and low-energy conformation with van der Waals surface of 

(a) theophylline; (b) CHT; (c) PD81,723; and (d) 2f. 

IV. CONCLUSION 

The possibility of allosterically modulating receptors offers novel pharmacological 

means of ‘‘fine-tuning’’ receptor function. Further clarification is 

required with respect to whether such modulated receptors are a general 

feature of all or only of a subset of GPCRs and whether endogenous agents 

regulate via this mechanism receptor function in vivo. Finally, elucidation 

of the molecular mechanisms of the allosteric interactions will provide 

useful insights for the therapeutic exploitation of this phenomenon in the 

design and development of appropriate modulatory drugs. 

Abbreviations 

ACh Acetylcholine 

cAMP Cyclic-3V,5V-adenosine monophosphate 

[ 3 H]CCPA [ 3 H]-2-Chloro-N 6 -cyclopentyladenosine 

CHO Chinese hamster ovary 

CH3CN Acetonitrile 


CHT 8-Cyclohexyltheophylline 

CMB N-Chloromethyl brucine 

CPA N 6 -Cyclopentyladenosine 

[ 3 H]DPCPX [ 3 H]-1,3-Dipropyl-8-cyclopentylxanthine 

CPT 8-Cyclopentyltheophylline 

DMF N,N-Dimethylformamide 

Et 3N Triethylamine 

Et 2NH Diethylamine 

GPCR G-Protein-coupled receptor 

HOAc Acetic acid 

NECA 5V-(N-Ethyl)-carboxamidoadenosine 

NMS N-Methyl scopolamine 

PD81,723 (2-Amino-4,5-dimethyl-3-thienyl)-[3(trifluoromethyl) 

phenyl]methanone 

QNB Quinuclidinylbenzilate 

R-PIA N 6 -[-(R)-1-Methyl-2-phenylethyl]adenosine 

Theophylline 1,3-Dimethylxanthine 


A. Kourounakis wishes to acknowledge support for this work from the 

European Commission financed programs BIO4-CT97-5138 and QLK6- 

CT-1999-51170. 

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9 

Protein Misfolding and 

Neurodegenerative Disease: 

Therapeutic Opportunities 

Harry LeVine III 

University of Kentucky, Lexington, Kentucky, U.S.A. 

I. DISEASES WITH PROTEIN MISFOLDING 

Alzheimer’s disease, a typically late-life dementia and the most prevalent 

chronic neurodegenerative disease, is pathologically characterized by the 

presence of insoluble h-amyloid peptide in extracellular senile and diffuse 

plaques and intracellular accumulation of neurofibrillary tangles (NFTs) 

of hyperphosphorylated tau protein as parahelical filaments. These 

insoluble protein assemblies are derived from normal cellular proteins 

that have deposited in entities that had been recognized histologically for 

some 70 years before their main protein constituents were determined. A 

potential unifying theme has emerged in the pathology of a number of 

chronic neurodegenerative diseases. Improved immunological and microanalytical 

methods have led to the identification of the constituents of 

other proteinaceous deposits associated with neurodegenerative disease. 

These deposits are intracellular, however, unlike those of the amyloid-beta 

(A h) peptides. A series of genetically dominant trinucleotide repeat 

diseases coding for glutamine in which the CAG repeats are expanded 

in a different protein in each disease was observed to develop insoluble 

polyglutamine-containing inclusions, concentrated in different brain areas 


and undergoing cell loss depending on the protein and disease involved [1– 

5] (see Table 1). The prototype is Huntington’s disease (HD) which affects 

the largest number of people. Polyglutamine stretches can form particularly 

stable h-sheet structures, which are prone to aggregation [6–9]. First 

described in transgenic mice overexpressing exon I of Huntingtin, which 

contains the polyglutamine repeat [10], nuclear inclusions of Huntingtin 

form in susceptible regions of the brain. The same ubiquitinated inclusions 

are also found in human HD tissue when appropriate antibodies are used 

[11–14]. Although the correlation of deposits with the synaptic and cell 

loss of the disease pathology is imperfect, similar to the situation in 

Alzheimer’s disease, their appearance is consistent with a pathway of 

protein folding and translocation that leads to cell loss. Similar observations 

have been made and conclusions reached for types 3 and 7 

spinocerebellar ataxia (SCA-3, SCA-7) and dentatorubral–palladoluysian 

atrophy (DRPLA), polyglutamine repeat diseases with cerebellar pathology 

[15–19]. A wide variety of insoluble proteins are associated with 

chronic neurodegenerative diseases (Table 2). Familial tauopathies, collectively 

referred to as FTDP17, are ascribed to mutations in various tau 

exonic or intronic sequences that alter mRNA isoform expression, resulting 

in insoluble fibrillar deposits of the microtubule-associated protein tau 

on human chromosome 17. Other tauopathies have been identified, 

varying with respect to the brain region affected and the ratio of the 

different tau gene splice products deposited [20]. Progressive supranuclear 

palsy and Pick’s disease are classic late-onset tau deposition diseases 

[21,22]. Tau is a conformationally ambiguous protein that does not adopt 

a defined structure in solution [23]. Hyperphosphorylation of tau favors 

conformational changes leading to rapid intermolecular h-sheet formation. 

This inhibits the microtubule-polymerizing activity of this microtubule-associated 

protein and leads to NFT formation [24]. The 

phosphorylation of a specific sequence on tau containing T231 facilitates 

binding and depletion of a prolyl isomerase, Pin1, effecting its nuclear 

function [25]. 

Prion diseases resulting in encephalopathy can be transmitted 

between individuals within species (more rarely between species) [26–28]. 

A conformational variant of the normal cellular protein PrP S (PrP C ) 

(protease-sensitive or cellular) is believed to catalyze [29] or nucleate [30– 

33] conversion to the pathological form, PrP R (protease-resistant). This 

highly unusual nongenetic mode of transmission of an infectious agent has 

been strongly debated [29]. The observation of multiple examples of 

nucleated catalysis of aberrant polymerization of protein subunits has 


Table 1 Polyglutamine (CAG Repeat) Neurodegenerative Diseases 

Disease Sites of pathology 

Repeat number 

Normal Disease 

Protein 

Location of 

disease (normal) a 

Huntington’s 

disease 

Striatum (medium, spiny) 11–34 36–121 Huntingtin NI (c) 

Spinocerebellar Cerebellar cortex (Purkinje cells), 6–39 40–81 Ataxin-1 NI (n, c) 

ataxia (SCA), type 1 brain stem 

SCA2 Cerebellum, pontine nucleus, substantia 

nigra 

15–29 36–64 Ataxin-2 NI (c) 

SCA3 (Machado– Substantia nigra, globus pallidus, 13–42 61–84 Ataxin-3 NI (c) 

Joseph disease) pontine nucleus, cerebellar cortex 

SCA6 

SCA7 

Cerebellar and mild brain stem atrophy 

Photoreceptors and bipolar cells, 

cerebellar cortex, brain stem 

4–18 

7–17 

21–30 

37–130 

VDCCa1Asubunit 

Ataxin-7 

NI (memb) 

NI (n) 

Spinal and bulbar 

muscular atrophy 

(SBMA) 

Motor neurons, dorsal root ganglia 11–34 40–62 Androgen receptor NI (n) 

Dentatorubral- Globus pallidus, dentatorubral and 7–35 49–88 Atrophin-1 NI (n) 

palladoluysian 

atrophy 

(DRPLA) 

subthalamic nuclei 

a NI, nuclear inclusions; (c), normal cytoplasmic localization; (n), normal nuclear localization; (membr), normal membrane localization. 


Table 2 Neurodegenerative Diseases with Insoluble Deposits 

Disease Sites of pathology Major protein Deposit 

Alzheimer’s disease Neocortex, hippocampus h Peptide; 4R, 3R tau Diffuse and senile plaques, 

paired helical formation, NFT 

Multiple=system tauopathy Frontotemporal regions, 4R tau NFT in oligodendroglia and 

(familial) 

brain stem, spinal cord 

neurons 

Progressive supranuclear 

palsy (PSP) 

Frontotemporal regions 4R tau NF NFT in astrocytes and neurons 

Corticobasal degeneration 

(CBD) 

Frontotemporal regions 4R tau NFT 

Pick’s disease Frontotemporal regions 3R tau NF Paired helical formation, NFT 

Diffuse Lewy body 

Cerebrocortical regions, a-Synuclein Lewy bodies and neurites 

disease (DLB) 

substantia nigra 

Parkinson’s 

Substantia nigra, brain a-Synuclein Lewy bodies and neurites 

disease 

nuclei 

Multiple-system 

atrophy (MSA) 

Cerebellum, striatal regions a-Synuclein Lewy bodies and neurites 

Amylotrophic 

lateral sclerosis (ALS) 

Brain stem, spinal cord a-Synuclein Neuronal cytoplasm 

Familial ALS Brain stem, spinal cord SOD1 mutants Neuronal cytoplasm 

Creutzfeldt–Jakob 

disease (CJD) 

New variant CJD 

Gerstmann–Straussler– 

Scheinker disease 

Fatal familial insomnia 

Kuru 

Prion protein Extracellular deposits 


markedly decreased the heretical flavor of such concepts. Transmissible 

protein aggregation has also been observed with [URE3], the prion form 

of Ure2p, a nonchromosomal genetic element regulating nitrogen catabolism, 

and with [PS1], the prion form of Sup35p in Saccharomyces 

cerevisiae [31,34]. 

The specific etiology of prion diseases in mammalian systems depends 

on the modified form of the protein [35], of which different variants 

display distinguishable conformations [36,37]. Most forms lead to a 

spongiform encephalopathy with marked neuronal cell loss in regions that 

accumulate the pathogenic protease-resistant conformer of the protein. 

Some of the more virulent forms of the protein expressed in Creutzfeldt– 

Jakob disease (CJD) are accompanied by classical intracellular amyloid 

plaques of PrP R . Although primarily recognized as a rare animal disease 

(scrapie), its appearance in the English beef herd in the 1990s and its 

potential for transmission to humans after a long latency caused a flurry of 

interest in detection and treatment countermeasures [38]. 

a-Synuclein, a synaptic protein, is deposited in Lewy bodies and 

Lewy neurites in Parkinson disease [39–41], in diffuse Lewy body disease 

[42], and in the Lewy body variant of Alzheimer’s disease [43]. Multiplesystem 

atrophy is characterized by intracellular neuronal and glial asynuclein 

inclusions [44]. The role for a-synuclein in these diseases was 

supported by the discovery of mutant forms of a-synuclein, A53T and 

A30P, in familial early-onset Parkinson’s disease [40]. Like tau, a-synuclein 

is a conformationally ambiguous protein with little stable secondary 

structure in solution [45]. a-Synuclein, but not the related h-org-synuclein, 

can polymerize in a nucleation-dependent fashion [46–48]. 

Lou Gehrig’s disease (amyotrophic lateral sclerosis: ALS) displays 

motor neuron deposits of hyperphosphorylated neurofilament subunits in 

the sporadic disease. Familial ALS, some 20% of all cases of ALS, involves 

dominant superoxide dismutase SOD1 mutants that can form h-barrel 

aggregates [49–51]. 

To this list of protein misfolding diseases can be added rare familial 

amyloidoses in which the mutated proteins have the classic amyloid fibril 

congophilic birefringence and cross-h-sheet structure (Table 3). Many of 

these deposits have an impact on the central nervous system (TTR, 

cystatin, lysozyme) as well as on other organ systems. A newly described 

disease, familial British dementia, is associated with the deposition of Abri, 

a 34 amino acid, 4 kDa peptide cleaved from a 277 amino acid precursor 

sequence, the last 10 amino acids of which are not normally translated [52]. 

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is 


Table 3 Amyloidoses Recognized by the WHO International Nomenclature 

Committee on Amyloidosis 

Precursor protein Associated disorder 

Immunoglobulin light chain Plasma cell disorders 

Immunoglobulin heavy chain Plasma cell disorders 

Apo-serum amyloid A protein Inflammation-associated, familial 

Mediterranean fever 

Transthyretin Familial amyloidotic neuropathy, 

systemic senile amyloidosis 

h2-Microglobulin Dialysis-associated amyloidosis 

Apolipoprotein AI Familial amyloidotic neuropathy, 

aortic amyloidosis 

Gelsolin Familial systemic amyloidosis 

Lysozyme Familial systemic amyloidosis 

Fibrinogen a chain Familial systemic amyloidosis 

Cystatin C Familial cerebral hemorrhage 

h-Amyloid precursor 

protein 

with amyloidosis 

Sporadic and familial 

Alzheimer’s disease, 

familial cerebral hemorrhage 

with amyloidosis 

Prion protein Spongiform encephalopathies 

Procalcitonin C-cell thyroid tumors 

Islet amyloid polypeptide Insulinoma, type II diabetes 

Atrial natriuretic factor Atrial amyloidosis 

Prolactin Prolactinomas; pituitary 

amyloidosis 

Insulin Iatrogenic amyloidosis 

Lactoferrin a 

Corneal amyloidosis a 

a Preliminary, awaiting confirmation by WHO International Nomenclature Committee 

on Amyloidosis. The term amyloidosis is reserved by the committee specifically for 

extracellular protein deposits. 

another rare hereditary dementing disorder resulting from point mutations 

in the neuroserpin gene [53]. FENIB is marked by unique neuronal 

inclusion bodies consisting primarily of abnormal aggregated neuroserpin 

filaments formed by a mechanism similar to that found in other familial 

diseases of serpin conformation, including emphysema and cirrhosis due to 

mutant a1-antitrypsin or thromboembolytic disease in antithrombin 

mutants [54]. 


The mechanisms of cell loss in these various diseases of protein 

deposition may differ in detail, but the association of insoluble protein 

inclusions with the pathology suggests that interventions preventing 

protein misfolding and deposition may be of therapeutic utility. Alternatively, 

stabilization of toxic species must be avoided, since the insoluble 

form of the protein is one potential strategy for reducing the exposure to 

toxic, soluble forms. Approaches similar to those applied to blocking Ah 

fibril formation in Alzheimer’s disease may prove fruitful with these other 

proteins, possibly extending to some of the same compounds being 

developed for AD. There are examples of this for prions [55–58] and for 

transthyretin [59]. 

The purpose of this chapter is to conceptualize the shared molecular 

features of protein misfolding in neurodegenerative diseases. By stressing 

the commonalities, rational strategies can be devised to target similar 

pathways that lead to cellular degeneration and eventually to clinical 

symptoms in these diseases. This is one way to maximize the effects of 

progress made for the pharmaceutically attractive (relatively large patient 

base) neurodegenerative diseases such as Alzheimer’s and Parkinson’s for 

application to other serious but less prevalent neurodegenerative diseases. 

Such ‘‘piggyback’’ strategies may be a starting point for therapeutics that 

already have the appropriate bioavailability, brain penetration, and longterm 

safety profile required for these applications. Similarly, nonneural 

amyloid diseases and diseases with significant amyloid components such as 

type II diabetes could also be approached. 

II. MECHANISMS OF PROTEIN POLYMERIZATION 

Protein homopolymerization is a well-studied process by which cellular 

structure is dynamically regulated in response to the environment and 

cellular metabolism. Actin and tubulin exist as nucleotide-dependent 

(ATP and GTP, respectively) polymers (microfilaments and microtubules) 

that rapidly elongate and shorten in a reversible manner 

regulated by binding proteins that can catalyze either polymerization 

or filament shearing. Mathematical analysis of the physical chemistry 

of the polymerization of these systems has defined the nucleation and 

elongation processes and provided the theoretical basis for models 

describing fibril assembly [60–63]. Nature has also provided evidence 

that small molecules, such as plant alkaloids and fungal secondary 

metabolites, are capable of modulating protein–protein interactions 


(actin depolymerization: cytochalasin, podophylotoxin; tubulin depolymerization: 

vinca alkaloids; microtubule stabilization: taxol). Another 

extremely physiologically important protein polymerization reaction that 

has been studied quantitatively is the process of thrombin-catalyzed 

fibrinogen fragmentation and assembly of fibrillar fibrin during the 

clotting of blood [64–66]. The larger size of the monomeric protein units 

in these polymers has simplified detection of the various intermediates 

in the assembly process. Atomic level structural resolution of these relatively 

large proteins has been aided by use of the modulators of 

polymerization [67–69]. 

Protein polymerization can also lead to pathological consequences. 

In contrast to physiologically normal assemblies, the pathological polymers 

are usually poorly reversible or degradable and tend to accumulate 

until they cause problems for the surrounding cells or tissue. The polymerization 

of mutant hemoglobin S inside the red blood cells of individuals 

afflicted with sickle cell anemia occurs rapidly and is modulated by the 

hemoglobin ligand 2,3-diphosphoglycerate. The mutation decreases the 

stability of the deoxygenated form of the protein, leading to exposure of 

hydrophobic surfaces and an increased propensity to aggregate. A model 

envisioning heterogeneous nucleation along the sides of the polymer and 

branching reactions in addition to the standard homogenous nucleation 

observed at the ends of growing fibrils was first described for the sickle cell 

hemoglobin system [70,71]. The effectiveness of hydroxyurea treatments in 

reducing the severity of the sickle cell crisis is ascribed to stabilizing effects 

on the mutant hemoglobin conformation [72]. 

Several pathological self-polymerizing systems have been biophysically 

characterized sufficiently to permit identification of protein or peptide 

species that could serve as molecular targets in a structure–activity 

relationship. These include transthyretin (TTR) [73–76], serum amyloid 

A protein (SAA) [77], microtubule-associated protein tau [78–80], amylin 

or islet amyloid polypeptide (IAPP) [81,82], IgG light chain amyloidosis 

(AL) [83–85], polyglutamine diseases [9,86], a-synuclein [47,48] and the 

Alzheimer’s h peptide [87–96]. A variety of Ah peptide assay systems 

have been established at Parke-Davis to search for inhibitors of fibril 

formation that could be therapeutically useful [97]. 

In the search for fibril formation inhibitors, the self-association to 

form amyloid fibrils of the Ah peptides containing 40 and 42 amino 

acids can be treated as a coupled protein folding and polymerization 

process passing through multiple intermediate peptide species. The in 

vitro challenge is (1) to identify the various conformational forms and 


multimeric species involved, (2) to establish their order and arrangement in 

parallel and/or in sequential pathways in a reaction scheme, and (3) to 

design assay conditions under which only one intermediate is rate limiting 

so that a reasonable structure–activity relationship can be determined. The 

in vivo relevance of a particular mechanistic scheme will eventually be 

assessed by the activity of bioavailable and brain-penetrant inhibitors of 

the defined in vitro reactions in in vivo transgenic models of central 

nervous system Ah amyloidosis. 

III. INTERMEDIATES IN FIBRIL FORMATION 

By analogy to the well-characterized polymerizing tubulin and actin 

protein systems, and consistent with experiment, h-peptide aggregation 

in the test tube is envisioned as nucleation event rate-limited by the 

formation of a multimeric intermediate from the monomeric random coil 

peptide in solution. Similar qualitative kinetics hold for light chain (AL) 

amyloid, IAPP, TTR, prion protein, tau, polyglutamine diseases, and asynuclein. 

Figure 1 suggests a schematic view of the process for the hpeptide 

separated into prenucleation, nucleation, and fibril growth phases. 

The techniques used to characterize peptide species at the different stages 

of the reaction are noted below the relevant intermediate in the figure. The 

presence of the early intermediates has been inferred from the kinetics of 

Figure 1 Stages of amyloid aggregation: steps in protein polymerization and the 

techniques used to measure them for the Alzheimer’s h peptide. 


Ah fibril formation; an identifiable nucleating species has yet be isolated. 

Direct observation has been made difficult by the small size of the h 

peptide, which has an effective hydrodynamic radius of 4 nm [98–100], and 

by the apparent low abundance of nucleating species due to the low 

probability of their formation. Such species would be formally akin to 

an enzyme transition state that is usually kinetically inferred or sometimes 

trapped with certain kinds of inhibitor. In disaggregated, ultrafiltered 

(20 nm pore size) preparations, less than 1% of the molar peptide concentration 

is inferred to be present as ‘‘seeds’’ or nuclei determined by the 

kinetics of fibril formation [101]. 

There is, however, ‘‘hard’’ evidence for the involvement of transient 

species in h-peptide fibril formation. Recent atomic force microscopy 

(AFM) [93,94,102–104] and electron microscopic observations [105,106] 

have characterized rope like species intermediate between nucleation and 

fibril extension. Designated as protofibrils, these species appear to anneal 

and to wind around each other. Such a model is consistent with oriented 

x-ray fiber diffraction patterns of a triple or higher helix of h sheets 

producing a h-helix quaternary fibril structure [107–113]. These protofibrils 

are on-pathway intermediates in amyloid fibril formation [93,94] 

containing h-sheet structure. They are negative with respect to thioflavin 

T and apparently toxic to cultured cells [105]. A stiffer, more compact 

fibril species (type I) is eventually formed from the initial type II fibril, 

10–20 nm high. The dominant fibril form observed is dependent on the 

environmental conditions and the initial conformational state of the 

peptide [103]. Recent AFM studies indicate that amylin fibrils grow 

bidirectionally, from both ends at roughly equal rates [114]. Branched 

fibrils and heterogeneous catalysis along the edge of the fibrils [70,71] 

have also been observed [94]. 

The variety of structures of h-peptide species observed by electron 

microscopy and by AFM suggest that different surfaces would be available 

to bind inhibitors on each species; moreover, the ability of a given inhibitor 

to block the fibrillization reaction should depend on the peptide species 

present in a particular situation. The rate-limiting species in vivo is 

unknown at present. It is also possible for more than one fibrillization 

pathway to operate concurrently, depending on the in vitro and in vivo 

reaction conditions. A host of molecules have been claimed to inhibit hpeptide 

amyloid fibril formation on the basis of a variety of assays for 

activity. The diversity of structures is represented in Figure 2. Their efficacy 

is in general low (IC 50 tens of micromolar or higher), corresponding 

roughly to the order of magnitude of the amount of peptide present in 


Figure 2 Reported inhibitors of Ah aggregation: 1, nicotinamide [156]; 2, Anthranilates [59]; 3, N-alkyl-N-methylpiperidinium bromides 

[157]; 4, benzothiazoles [U.S. patent 6,001,331]; 5, Congo Red [142]; 6, melatonin [158]; 7, PPI-558 (Praecis Pharmaceuticals, Inc. patent 

WO 9628471); 8, anthracyclines (IDOX) [159]; 9, aza-anthracyclones (WO 9832754-A); 10, iminoaza-anthracyclinones (WO 9832754-A); 

11, acridinones (U.S. patent 5,972,956); 12, naphthyl monoazo compounds [U.S. patent 5,955,472]; 13, porphyrins [160]; 14, 

naphthalenes (Japanese patent 090954222, Teijin KK); 15, rifamycins [161]; 16, rifampicins [161]; 17, alkylsulfonates/sulfates [162]. 


most assays, implying a 1:1 compound-to-peptide stoichiometry. In most 

cases, however, the stoichiometry for inhibition has not been determined. 

A high concentration of Ah peptide is generally used to overcome the 

unfavorable kinetics of multiple peptides interacting to form a nucleus 

capable of supporting the addition of monomeric peptide. Such reactions 

exhibit a lag phase until the nucleus is formed (Fig. 3, curve c). Inhibitors 

can affect either the lag phase, the maximal extent of the reaction, or both 

(Fig. 3, curve d). Unless both the nucleation and extension reactions are 

monitored, inhibitors prolonging the lag phase are poorly distinguished 

from those blocking extension from the nucleus, thus muddying any 

structure–activity relationships. Quantitative treatment of the reaction 

has been proposed to mathematically separate the nucleation and extension 

reactions [62,115]. Distinguishing true nucleation from various exponential 

growth mechanisms is actually quite difficult, requiring precise rate 

Figure 3 Effect of seeding and inhibitors on aggregation reaction. The lag phase 

(curve c) is characteristic of reactions in which formation of nuclei for 

polymerization is an unfavorable process. Addition of preformed nuclei or 

‘‘seeds’’ (curve a) abolishes the lag phase. Inhibitors may affect the formation of 

nuclei and influence either the lag phase, the extension of the nuclei changing the 

growth phase, or both (curve d). The inhibitor example (curve d) acts more 

strongly at nuclei formation than on the slope or plateau level of the growth phase. 


determinations of the first 10% of the reaction over a range of reactant 

concentrations [116]. Such measurements have not been achieved with the 

h peptide. 

An extension reaction from a nucleus (Fig. 3, curve a) is pseudo–first 

order in peptide concentration and thus more easily analyzed. Inhibitors of 

extension would be expected to decrease the reaction rate and/or extent 

(Fig. 3, curve b). The nature of the nucleus, however, is a variable as well. 

Preformed fibrils can act as one kind of nucleus, adding monomers at the 

ends or laterally, which can give rise to more complex kinetics [70]. 

Branching vs linear addition reactions can be distinguished in some 

situations by dynamic light-scattering methods. Another type of nucleus 

seems to be present in solubilized aqueous peptide solutions that can pass 

through a filter having a pore size of 200 nm, but not 20 nm [101] and 

displays linear kinetics of fibril extension [117]. These species are present in 

too low an abundance to be observed directly, though they are detectable 

kinetically. 

Distinguishing the preformed and endogenous nucleus forms is 

problematic. The behavior of the accretion of soluble peptide onto AD 

plaques in tissue sections [118] or onto sonicated fibrils [119] is kinetically 

similar to that of spontaneous soluble nuclei. However, the endogenous 

soluble nuclei are not equivalent on a molecular level to fibril or AD plaque 

nuclei, since molecules such as Congo Red inhibit endogenous soluble 

nuclei extension at 0.25 AM [97], while over 700 AM is required to block 

accretion of monomer h peptide onto fibrils or plaques [119]. An assemblycompetent 

form of the h peptide, h10–35, has been shown to interact with 

fibrils and plaques [120] and to adopt a particular conformation in solution 

as determined by NMR spectroscopy [121], although no high affinity 

inhibitors of this accretion reaction have been reported. Alternatively, 

low abundance conformational forms of monomeric peptide may be the 

actively associating form of the peptide to endogenous seeds, accounting 

for the high (micromolar) amounts of peptide required in vitro for the 

extension reaction. These forms may be more abundant in biological 

systems, allowing fibril formation to occur at the low bulk concentrations 

(nanomolar) of Ah peptides found in vivo. 

As a result of the confusion over the identity of nucleating h-peptide 

species, prenucleation events remain poorly defined. A variety of methods 

possessing different degrees of resolution have been employed to look at 

these early stages in fibril formation. Chemical [122] and enzymatic 

(transglutaminase) [123,124] cross-linking, electron microscopy (EM) 

[105,106,125], AFM [93,94,102,103], ultracentrifugation, dynamic light 


scattering, and fluorescence resonance energy transfer (FRET) with modified 

beta peptides [89,105] have probed the oligomerization process 

preceding nucleus formation but have not yielded definitive structural 

information on the species present or the extent of their participation in 

nucleus formation. Difficulties arise from the small size (4.3 kDa) of the 

monomeric peptide unit, the simultaneous presence of multiple species of 

peptide, both conformational and association states, and their transient 

nature (since they rapidly form amyloid fibrils as their concentrations 

increase). As specific inhibitors of early stages in fibril growth are discovered, 

peptide species will be better defined, particularly if the intermediates 

can be trapped and their structures determined. 

Fibril extension from nuclei preformed under defined conditions 

has been characterized through a series of nucleus-dependent kinetic 

assays. The process of fibril formation from a nucleus in equilibrium with 

soluble, mostly monomeric peptide has proved much more amenable to 

study than the formation of the nucleus itself. Fibrillar species are readily 

detected by growth in size (filtration, sedimentation, static light scattering–turbidity), 

amyloid-specific reactivity with the optical probes Congo 

Red and thioflavin S and T, and by EM and AFM. Endogenous soluble 

nuclei or seeds form in aqueous solution, accumulating slowly at low 

temperature. Brief treatment with denaturants, organic solvents, and 

treatment with neat trifluoroacetic acid (TFA) or concentrated formic 

acid breaks down these seed structures, restoring the lag period of 

unseeded fibril formation. 

The processes of both seed formation and fibril extension are 

dependent on temperature and on peptide concentration, with 37jC 

being required for establishing equilibrium within 24 h with 30 AM 

h1–40. A full description of the assay system may be found elsewhere 

[97,117]. A 4 h reaction time is typically within the linear portion of the 

time course. This nucleus-dependent assay detects mainly inhibitors that 

are substoichiometric with the monomeric peptide, which is present at 

high concentration. It is relatively insensitive to inhibitors that target 

the monomeric peptide. Whether the inhibitors interact with the growing 

end of a seed or with a low abundance conformational form of the 

h peptide that is competent to add to the seed is difficult to determine at 

this time. Similar dose–response curves are obtained for Congo Red as 

an inhibitor with either thioflavin T (ThT) fluorescence or filtration of 

radioiodinated peptide readouts (Fig. 4) Caveats in the interpretation of 

both the ThT and radiometric filtration assays for the evaluation of putative 

inhibitors are discussed elsewhere [97]. 


Figure 4 Inhibition of nucleated fibril extension by Congo Red: h1–40 fibril 

formation detected by filtration of either radiolabeled peptide (circles) or 

thioflavin T (ThT) reactivity, (triangles) is inhibited by Congo Red with similar 

potency. For assay details, see Ref. 97. 

The prediction that fibrillization reactions proceeding via different 

folding pathways governed by different rate-limiting steps could be 

subject to different modes of inhibition appears to be substantiated. 

The endogenously seeded type of assay identifies types of inhibitor 

different from unseeded assays by using light scattering or turbidity 

detection. With the exception of the naphthyl monoazo benzo compounds 

(12) and the acridinone series (11), the molecules reported in 

Figure 2 are ineffective (IC 50 > 100 AM) in the presence of 30 AM 

h1–40 in seeded assays. In particular, short peptide sequences derived from 

the h16–25 amyloidogenic core of the h peptide KLVFFA are ineffective 

under the seeded assay conditions, although many modifications 

of this sequence have been studied [126–129], some of which (e.g., 7) 

are being developed as therapeutics. Inhibitors effective in the seeded 

assay format such as Congo Red are inactive in an accretion assay 

onto immobilized fibrils [119]. Rifampicin and daunomycin are very 

weakly active against accretion [130]. 


IV. CELLULAR SYSTEMS AND AMYLOID FIBRILS 

One of the reported biological effects of h-amyloid and amylin fibrils 

is cellular toxicity, inferred in vivo and modeled in various tissue 

culture systems. While amyloid fibrils were initially thought to be the 

toxic species, it has become increasingly clear that some other entity, 

probably soluble oligomers of the h peptide [131,132] that are in 

equilibrium with fibrils, are the culprit. Thus, in developing aggregation 

inhibitors that would be therapeutically useful, it is important to 

demonstrate that the nonfibrillar peptide species stabilized by inhibitor 

treatment are not toxic to cells. The selection of an appropriate 

cellular system is important because the resistance of cell types to 

the toxic effects of the Ah peptide varies significantly, often requiring 

industrial (50–100 AM) concentrations of peptide or the use of the 

nonbiological h 25–35 fragment. Mixed neuronal/glial or pure neuronal 

embryonic hippocampal or cortical cultures would seem to be the most 

relevant cell type, since neuronal cell death and dysfunction are 

hallmarks of neurodegenerative disease like AD. Unfortunately, the 

embryonic primary cultures are irregularly resistant to the effects of 

h1–42 when cell death is monitored. These cultures are heterogeneous 

mixtures of neuronal cell types, only some of which seem to be 

affected by the Ah peptide. In addition, embryonic mouse neurons 

are not the same as the deeply differentiated cells in the brain of an 

80-year-old human. Cultured PC12 cell lines have become a favorite 

system, with changes in MTT formazan production serving as a 

readout. However, the formazan deposition is not related to cell 

survival [133–136] and so is not reliable as an indicator of the effects 

of amyloid on cell death. 

Another prominent site of deposition of h-amyloid fibrils with age 

and in AD is within the cerebrovasculature in areas of the brain prone 

to parenchymal amyloid deposition [137–139]. The peptide deposits 

along the surfaces of the smooth muscle cells of the vascular wall, 

resulting in the death of those cells and their replacement by amyloid 

fibrils, weakening the vascular wall. Endothelial cells are also affected 

[140]. The ‘‘Dutch’’ mutation in the APP precursor protein Q22E, 

within the h-peptide sequence, produces a particularly fibrillogenic 

and toxic (to smooth muscle cells) peptide associated with primarily 

vascular deposition of mutant peptide and hemorrhagic vessel disease 

[137]. Thus, in addition to neuronal cells, the brain vascular smooth 

muscle cells are a pathologically relevant cell type. While the source of 


the h peptide in these deposits (brain or smooth muscle cells) is under 

debate, the smooth muscle cells in culture generate prodigious amounts 

of h peptide and accumulate C-terminal fragments of hAPP [139]. 

Organotypic cultures of the leptomeningeal blood vessels will accumulate 

exogenously applied, fluorescently labeled Ah peptide [141]. The 

leptomeningeal vascular smooth muscle cells (VSMC) isolated from 

either human or canine sources have proved to be reliable indicators for 

h-amyloid toxicity. Overnight treatment with 10 AM h 1–42 leads to 

deposition of fibrillar peptide in ThS-positive strands onto the cell 

surface and apoptosis of 70 to 80% of the VSMC assessed by 

bisbenzamide staining of condensed nuclei. For this cell type, preformed 

fibrils have little effect on cell survival, and the added fibrils remain 

scattered over the surface of the culture dish. 

Interpreting the effects of amyloid-modulating compounds on 

h 1–42-induced cellular toxicity and relating the results to in vitro aggregation 

inhibition is far from straightforward. A number of compounds 

are toxic to cells by a variety of routes. Besides interfering with 

aggregation, test compounds can block binding to the cell surface, 

internalization of h peptide, or any of a myriad of cellular events that 

could affect expression of h-peptide toxicity. Lack of effect of a 

compound could indicate that it is not blocking the toxic ‘‘site’’ on 

the peptide species, that it is not penetrating the cell, or simply that 

the compound is adsorbed, sequestered, or metabolized to an inactive 

form. In the VSMC system as in other cellular systems [142], Congo 

Red blocks both aggregation on the cell surface and h1–42 toxicity at 

10 AM, roughly equivalent to the total added peptide concentration. 

For optimal effect it must be added either before or along with the h 

peptide. Since the IC50 for an antiaggregation effect on 30 AM peptide 

in vitro is 0.25 AM, nonspecific adsorption of the compound to 

cellular components and to h-peptide fibrils may be mitigating the 

effects. Congo Red and other polysulfonate/sulfate polyanions are 

known to displace proteins from binding sites on the cell surface 

[143]. Congo Red’s practical therapeutic potential is limited because it 

does not penetrate the cell membrane or the blood–brain barrier, and 

the azo linkages are susceptible to metabolism and carcinogenic 

liability. Such difficulties can be addressed by structural modifications 

in inhibitors that will likely also improve some of the pharmacokinetic 

properties in vivo. However, the connection between cellular effects 

and desired in vivo properties of bioavailability and brain penetration 

is also not straightforward. 


V. ANIMAL MODELS OF BRAIN DEPOSITION 

OF INSOLUBLE PROTEINS 

The lack of animal models of Alzheimer’s disease that faithfully 

reproduce the human condition has significantly retarded development 

of therapies that would be expected to have a significant impact on 

the disease or its progression. Such models would provide a test bed 

for increasing confidence that a given therapeutic regime would have 

the desired effect in animals and would give insights that only 

experimental manipulation can provide into the disease process. 

Cholinomimetic approaches employed models of cholinergic deficit 

to bring the current anticholinesterase drugs to market, even though 

they only modeled a subset of the pathology of AD [144]. Serious 

discovery and development of therapeutic agents directed at the 

deposition of h-amyloid fibrils was put on a firm footing by the 

development of mice that overexpressed human APP, and depicted 

fibrillar and nonfibrillar h1–40 and h1–42 in the appropriate brain 

regions. Various hAPP mutants responsible for early-onset familial 

AD have been the most effective, particularly in combination with 

transgenic presenilin mutants, another familial AD locus. For a review 

of the hAPP mouse and other brain amyloidosis models see Walker [145]. 

Again, these are only partial models of AD, since no significant neuronal 

cell death has been observed with these mice. Importantly, the Hsiao 

mouse, Tg 2576 (human APP695, Swedish mutation under the control of 

the mouse prion promoter), has been available to both commercial and 

academic laboratories, and thus there is a considerable shared pool of 

information and experience with this model. Direct comparisons with the 

other hAPP mice are limited because those models are not generally 

available. The Hsiao mouse shows robust and increasing deposition of h 

peptide from age 9 months onward, as well as dystrophic neurites, reactive 

astrogliosis, microglial activation around senile plaques, and some phosphorylated 

(but not tangled) tau immunoreactivity, but no detectable 

neuronal cell death or reduction in synaptic counts, despite plaque 

densities approaching that of clinical AD. 

An important validation of the mice that overexpress human mutant 

hAPP as a platform for testing therapeutics targeting h-peptide deposition 

has been provided by the Elan company, using their PDAPP mouse [146]. 

Immunization of the mice, either at an early age or after plaques had 

formed, resulted in clearance of immunoreactive plaques and peptide from 

the subjects’ brains. Although the elucidation of the mechanism explaining 


this potential therapy has not been reported, the experiments establish that 

deposition of the peptide can be interrupted, and even reversal of preformed 

plaques is possible in this type of animal model. Human trials of the 

safety and efficacy of the immunization protocol are to start in the near 

future. The plaques are thus dynamic structures, and therapeutics that 

interfere with Ah deposition or production would be predicted to reduce 

brain h-amyloid load [147]. 

Similar partial neurodegenerative disease animal models involving 

insoluble protein deposition have been developed for Huntington’s 

disease [148], spinocerebellar ataxia type 1 (SCA1) [149,150], and 

Machado–Joseph disease (MJD; SCA3) [151], all trinucleotide repeat 

disorders. The protein deposits, consisting primarily of the polyglutamine 

tract, accumulate intracellularly, eventually collecting in the 

nucleus, where they are thought to disrupt nuclear function both in 

humans and in the animal models. As for Alzheimer’s disease and the 

Ah peptide and tau proteins, there is considerable controversy about 

the relevance of the deposits to the disease process because observed 

neuronal cell loss does not overlap entirely with visible inclusions. The 

arguments in favor of relevance are that the toxic species may be 

smaller oligomeric species not observed by microscopy and that in some 

situations the deposits may serve a protective function by sequestering 

potentially toxic material. 

The Huntington’s disease models have been studied in considerable 

detail. The pathology bears a striking resemblance to the human disease in 

a number of respects, although polyglutamine overexpression is not a 

complete model. The Bates R6 mice expressing exon I of the human 

Huntingtin protein, consisting of the N-terminal 17 amino acids + a 

pathological number (115–156 CAGs) of glutamines + 52 more amino 

acids under control of the human promoter, develop age-dependent, brainregion-specific 

cell loss accompanied by nuclear inclusions and behavioral 

and motor abnormalities reflecting those in the human disease, leading 

eventually to death [152]. Time of onset, severity of the symptoms, and 

length of disease are dependent on the number of glutamine repeats in the 

observed human pathological range. A longer term mouse model with the 

full-length human Huntingtin under its natural promoter, which lacks 

the potential diabetic condition of the Bates mouse, may provide a more 

realistic picture of the HD process, although it would be less useful for 

rapid testing of potential therapies. 

Mice expressing high levels of human a-synuclein under the 

control of the human PDGFh promoter developed intracellular nuclear 


and cytoplasmic inclusions that were immunoreactive with antibody 

specific for the human a-synuclein [153]. As early as 2 to 3 months of 

age in the transgenics, inclusions were found in the deeper layers of the 

neocortex, the CA3 region of the hippocampus, the olfactory bulb, and 

occasionally the substantia nigra, regions typically affected in Lewy 

body disease. While nigral tyrosine hydroxylase–positive cell number 

was similar to nontransgenic littermates, TH-positive nerve terminals 

were significantly reduced, as were TH immunoreactivity and enzymatic 

activity in 12-month-old animals. In the 12-month-old mice, neurological 

impairment similar to that found in Parkinson’s disease was 

demonstrable in rotorod performance. 

Human prion disease models have also been developed in mice 

[154,155]. Crossing the species barrier into an experimentally accessible 

animal system, the prions responsible for Creutzfeldt–Jakob disease, new 

variant CJD, Gerstmann–Straussler–Scheinker disease, and fatal familial 

insomnia produce a reproducible time-dependent neuronal degeneration 

leading to death. 

VI. CLINICAL TRIALS FOR AD TESTING OF 

POSSIBLE DISEASE-MODIFYING AGENTS 

While testing of amyloid aggregation inhibitors against AD in human 

subjects is a way off, it is worth considering how such trials should be 

conducted to establish clinical efficacy. With all the genetic and 

biochemical evidence that the h peptide is implicated in AD pathology, 

it very well may not be the only relevant pathology in all patients for this 

very complex disease. It remains distinctly possible that removal of all 

amyloid plaques and/or h peptide from the brains of AD patients will 

not restore cognitive function in advanced stages of the disease. Thus, a 

paradigm treating significantly cognitively impaired patients to look for 

a leveling off in their decline or a reversal to normal may not show the 

desired treatment effect with an aggregation inhibitor. To be fair, it may 

not show effects with any treatment if too many neurons have died or 

become dysfunctional. The cholinomimetic therapies were symptomatic 

treatments designed to supplement function (acetylcholine) that had 

been lost without regard for the process that caused that functional 

loss. In attacking what is believed to be a fundamental process in 

disease progression, other measures may be needed to reverse the 

degeneration that has already occurred, assuming that not too much 


damage has been inflicted. Disease-modifying therapies are most likely 

to influence progression to disease and/or delay onset of symptoms. 

Proper assessment of the effects of a therapy will require clinical trial 

designs that make the appropriate measurements. It will be important 

to assess plaque load and/or h-peptide level in patients treated with 

aggregation inhibitors or other modes of reducing either brain hpeptide 

content or its effects in addition to the classical cognitive end 

points. Whatever the outcome of the trials, in interpreting the results 

for the development of new generations of therapeutics it is important 

to determine whether the therapy accomplished what it was designed to 

do—reduce amyloid peptide deposition. If h peptide is eliminated but 

no therapeutic benefit is observed, we should conclude that the h 

peptide is not the major player—at least in the patient population selected 

for study. The answer will be important in justifying future 

pharmaceutical investment as well as in guiding future research for 

effective therapies against AD. 

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10 

Uncoating and Adsorption Inhibitors 

of Rhinovirus Replication 

Guy D. Diana 

ViroPharma, Inc., Exton, Pennsylvania, U.S.A. 

Adi Treasurywala 

Pfizer Central Research, Groton, Connecticut, U.S.A. 


Rhinoviruses are responsible for approximately 50% of infections resulting 

in the common cold [1]. These infections are caused by over 100 distinct 

serotypes, which vary by exhibiting minor or major changes in 

structure. The virus is divided into a major group, consisting of approximately 

90%, and a minor group, differing by the mode of attachment of 

the virus to the cell. The members of the major group of serotypes have 

been shown to bind to domain 1 and 2 of ICAM-1 [2–5], while the minor 

serotypes appear to have a binding preference for the human low density 

lipoprotein receptor (LDLR) [6]. An effective antirhinovirus agent would 

be expected to be active against the majority of serotypes, since at any time 

one may become infected by any of the 100+ serotypes. The compounds 

in the series shown in Figure 1 have demonstrated broad spectrum antirhinovirus 

activity against both the minor and major group of serotypes 

[7–12]. These compounds have been shown to inhibit uncoating of the 

major group [13] and to block adsorption of the minor serotypes to the cell 

[14]. This chapter describes our efforts to determine the mode of binding 


Figure 1 General structure of isoxazole series of antipicornavirus agents. 

of these compounds and to enhance the activity by utilizing x-ray crystallography 

coupled with traditional structure–activity methodology. 

II. CAPSID BINDING COMPOUNDS 

In 1985 Dr. Michael Rossmann and his colleagues determined for the 

first time the three-dimensional structure of a human rhinovirus [15]. 

Their studies, performed with human rhinovirus type 14 (HRV-14), revealed 

the structure as an eicosahedron consisting of four proteins designated 

VP1, VP2, VP3, and VP4 forming a protomeric unit, combined 

to form a fivefold axis of symmetry (Fig. 2). The surface of the capsid 

Figure 2 The three-dimensional structure of HRV-114 consisting of four viral 

proteins, VP1, VP2, and VP3; Vp4 is pointing toward the center of the capsid 

protein and is not visible. 


Figure 3 The orientation of disoxaril in the binding site of HRV-14. 

protein contains a canyon that was shown to be the cell receptor binding 

site [2]. Subsequently, the structure of several additional rhinovirus 

serotypes was determined [16–19]. Although these rhinoviruses share 

the same general structure described for HRV-14, the latter appears to be 

distinctly different from other rhinoviruses, particularly with respect to 

the sequence similarity. Following the elucidation of the structure of 

HRV-14, x-ray studies were performed on two members of the series of 

compounds shown in Figure 1, disoxaril and WIN52084 [20]. The purpose 

of this study was to elucidate the nature of the binding of these 

compounds to the capsid protein. 

Disoxaril was shown to bind in a hydrophobic pocket below the 

a depression referred to as the ‘‘canyon,’’ with the oxazoline ring in the 

‘‘toe’’ region of the binding pocket. The isoxazole ring resides in the ‘‘heel’’ 

below the area designated as the pore (Fig. 3). The nitrogen of the isoxazole 

Figure 4 Structure of WIN52084. 


ing was within 3.5 A ˚ of asparagine 219, suggesting the possibility of 

hydrogen bonding. The phenyl ring was in a stacking conformation with 

tyrosine 128 and tyrosine 152. WIN52084, however, was bound in the 

opposite orientation, with the isoxazole ring in the ‘‘toe.’’ Subsequently, it 

was determined that only two additional compounds that were examined 

were bound in the same orientation as WIN52084 (Fig. 4) [21]. This 

observation led to the following conclusions: 

Analogues with a seven-carbon chain connecting the phenyl and 

isoxazole rings, and with a substituent on the oxazoline ring, 

were bound with the isoxazole ring in the toe of the hydrophobic 

pocket. 

All other analogues, regardless of the length of the connecting chain, 

were bound in the opposite orientation. 

A. The Nature of the Binding Site 

The influence of substituents connected to the oxazoline ring on the binding 

orientation of these molecules was intriguing. Since the carbon to 

Figure 5 Homologues of WIN52084 illustrating an entaniomeric effect. The 

asymmetric center on the oxazoline ring is designated by asterisk. 


which the alkyl substituents are attached is asymmetric, both enantiomers 

of WIN52084 as well as a homologous series of compounds were 

evaluated against HRV-14 [22] (Fig. 5). In each case, the S isomer was 

considerably more inhibitory than the R, which suggested an enantiomeric 

effect. Examination of WIN52084 in the pocket clearly showed that 

the S-methyl group was in close proximity to a hydrophobic pocket 

formed by Leu106 and Ser107 (Fig. 6). 

To further analyze the interactions of the methyl group of the two 

comformers in the binding site, an energy profiling study was performed. 

With the x-ray crystal structure of the S isomer of WIN52084 in the virus 

pocket serving as a starting point, a window consisting of all residues 

within 8 A ˚ of any atom was excised from the starting structure. After 

charges had been set on the atoms of the resulting pocket and drug 

according to a method in Chem-X [23], and after the hydrogen atoms 

had been removed, the intermolecular van der Waals energy was calculated 

via a 6–12 function for conformations resulting from the rotation 

of the oxazoline ring about the bond connected to the phenyl ring, 

in increments of 10j. A plot of this function versus the rotation angle 

Figure 6 WIN52084 bound to HRV-14. The methyl group on the oxazoline ring 

is pointing toward a hydrophobic pocket formed by Leu106 and Ser107. 


showed mainly two peaks at –90j and 100j. A repeat of this calculation 

with the R conformer resulted in a flat valley between 30j and approximately 

120j. Similar results were obtained with the R- andS-ethyl 

compounds, which showed an even more dramatic pattern that was 

significant because the ethyl homologue was more potent (Fig. 7). These 

results suggested that the twist angle about the two rings could be an 

important factor in determining biological activity. It is possible that 

the conformation with the appropriate twist angle may be imposed by 

the nature of the binding pocket and that maximum interaction with the 

hydrophobic pocket formed by Leu and Ser may also be of importance 

[20–22]. 

B. Aliphatic Bridge 

The x-ray studies on several analogues in this series of compounds showed 

that the chain connecting the isoxazole and phenyl rings adopts a bowed 

Figure 7 Plot of energy vs torsion angle from an energy profiling study resulting 

from rotating the oxazoline ring of the S isomer of WIN52084 about the phenyl 

ring. 


conformation when bound to HRV-14. It had been assumed that flexibility 

of the chain was critical for binding and biological activity. Dynamic 

studies by Dr. Andrew McCammon with WIN52084 in HRV-14 revealed 

considerable motion of the aliphatic chain during an observation lasting 

for 10 ps (Fig. 8) (Dr. Andrew McCammon, University of Houston, 

personal communication). This result posed several questions regarding 

the importance of flexibility vs rigidity of the chain. Would a conformationally 

rigid chain offer enhanced hydrophobic interactions and consequently 

improved binding, or are there other factors in the binding 

process that would require a flexible chain? To address these issues, several 

compounds with rigidity incorporated into the chain were synthesized; 

their activity against HRV-14 and HRV-1A examined (Fig. 9) and the 

compounds modeled in the respective binding site [24]. WIN54954, which 

Figure 8 Molecular dynamics of WIN52084 in HRV-14 during a 10 ps run, 

illustrating the movement of the chain. (Courtesy of Andrew McCammon, University 

of Houston.) 


Figure 9 Table comparing the activity of the E and Z olefin and butyne analogues 

of WIN54954. 

has been clinically evaluated, was used as a comparator. The Z olefin 

demonstrated a two- to threefold reduction in activity in comparison to 

WIN54954, while the E isomer showed a threefold enhancement in 

activity. The potency of the butyne analogue was more than fourfold 

greater than that of WIN54954 against HRV-14 and was comparable to 

that of the E isomer. 


C. Modeling of Conformationally Restricted 

Analogues 

The structures shown in Figure 9 were constructed using WIN54954 as a 

template, since its x-ray conformation in HRV-14 had been determined. 

The resulting structures were subjected to the Tripos force field (Maximin 

2), using Sybyl version 5.41, with default settings. Rotatable bonds in the 

alkyl ether chain were defined, and the structures were flexibly fitted to 

WIN54954, in virus-bound conformation, for insertion into the HRV-14 

binding site (Fig. 10). The optimized fitted structures were inserted into 

each serotype by replacement of virus-bound WIN54954. Since the drugbound 

conformation of the virus binding site with several of the compounds 

had been determined, revealing only minor variations in compound 

structure, insertion of the modeled compounds into the binding site 

configuration, derived from WIN54954, appeared reasonable. 

Two interesting observations emerged from this study. The acetylene 

analogue, which was more than fourfold more potent than WIN54954 

Figure 10 Overlay of energy-minimized structures of the E and Z isomers and 

WIN54954. 


against HRV-14, was inactive against HRV-1A, and there was a dramatic 

difference between the activities of the E and Z olefins against HRV-14. 

Because x-ray studies had shown that the HRV-14 binding site was longer 

than the 1A site, the results of this study supported the premise that the 

activity is dependent on the length of the molecule. The butyne was 

modeled in HRV-14, causing no serious steric interactions. However this 

was not the case in HRV-1A, where the chlorine atom appeared to interact 

with Ile125. 

The difference in activity of the E and Z olefins against HRV-14 was 

explained by examining the relatively low energy virus-bound conformations. 

The result of an overlay of WIN-54954 (based on x-ray crystallography 

data), minimize E- and Z-olefinic structures and the butyne 

analogue, suggested that the E isomer showed a reasonable fit while the 

Z isomer did not. Furthermore, when the Z isomer was inserted into the 

HRV-14 pocket, unfavorable interactions occurred. 

The very high minimal inhibitory concentration (MIC) values for the 

Z isomer against HRV-14 and HRV-1A may reflect a slow kon in both 

cases. The conformational space accessible to the isoxazole of the E and Z 

olefins, the butyne, and the three-carbon chained homologue of WIN 

54954 by conformational sweep graph and for the Z olefin disclosed a 

significant inaccessible region of space, while the butyne, E olefin, and 

alkane do not show this deficit. Consequently, binding to this site may be 

dependent on conformational permissibility in this region that is required 

for entry into the pocket. These results suggested that the activity of these 

compounds against the two serotypes is strongly dependent on the 

flexibility of the hydrocarbon chain and the ability of the molecule to fit 

into the conformational space of both pockets. 

III. PHENYL STACKING 

Thus far all the compounds that were examined bound to HRV-14, with 

the exceptions noted, are oriented with the phenyl ring in a stacking mode 

with Tyr128 and Tyr152. Aromatic–aromatic interactions have been 

shown to be quite common in protein–protein interactions [25–31], 

and in many cases have displayed [32,33] an electrostatic component. 

Furthermore, such interactions would be expected to contribute extensively 

to the binding energy [34]. To determine the nature of the aromatic 

stacking interactions, an energy profiling study was performed by twist- 


ing the phenyl ring about the carbon oxygen bond and examining a 

number of parameters such as heat of formation and electronic energy. 

Figure 11 shows the torsion angle between the oxazoline and phenyl rings 

for each compound after energy minimization and flexible fitting. These 

studies were performed with a variety of substituents in the position ortho 

to the ether. It was anticipated that if electrostatics were involved in the 

stacking of these rings, the correlation of the results of the profiling study 

with antiviral activity should relate to the physical and electronic properties 

of the substituents. There was no correlation between energy maxima 

or minima and size or electronic nature of the substituent, however, nor 

do the results correlate with biological activity. We concluded that electrostatics 

play no part in the stacking; rather, the interactions appear to 

be hydrophobic. In addition, these results suggest that a planar orientation 

of the phenyl rings is preferred. The lack of an electrostatic effect 

associated with the phenyl–phenyl interactions may be due to the inability 

of the phenyl ring of these compounds to adapt a true end-surface 

orientation as a result of space constraints within the pocket. 

Figure 11 Torsion angle between the oxazoline and phenyl rings obtained from 

minimized structures fitted to the x-ray structure of WIN54954. 


IV. HYDROGEN BONDING 

The initial x-ray study with disoxaril and WIN52084 in the binding site 

revealed that asparagine 219 was within hydrogen-bonding distance of the 

nitrogen of the isoxazole or oxazoline rings. Several pieces of information 

suggested that this type of binding contributes negligibly if at all to the total 

binding energy. Lau and Pettitt [35] examined whether the close approach 

of the asparagine and isoxazole ring, which had been observed crystallographically, 

was indeed an attractive event. By selectively computing the 

pairwise attraction of the hydrogen of the asparagine 219 and the nitrogen 

of the isoxazole ring, which could conceivably be involved in the hydrogen 

bond, and disregarding the contribution of this energy to the overall energy 

of the system, the researchers were able to predict that the potential 

hydrogen bond was inconsequential. 

In addition to the computational studies that argued against the 

existence of a hydrogen bond with Asn219, further evidence was obtained 

by site-directed mutagenisis of the asparagine in question to an alanine 

(Dr. Daniel C. Peaver, Sterling Winthrop Inc., personal communication). 

Confirmation of the mutation was accomplished by sequencing. A comparison 

of the sensitivity of the mutant with the wild type showed that no 

change in sensitivity had resulted from the removal of the hydrogen donor 

potential. Consequently, these findings were in complete agreement with 

the results reported by Lau and Pettitt. 

Although the evidence presented strongly suggests the lack of contribution 

of Asn219 to the binding energy, examination of the x-ray result 

of HRV-14-bound compounds revealed the presence of a water molecule in 

the vicinity of the isoxazole ring and hydrogen-bonded to the backbone of 

Leu106, Ser107, and Asn219 (Fig. 12) [36]. A similar hydrogen-bonding 

network has been seen in HRV-50 (Dr. Vincent Giranda, Sterling Winthrop 

Inc., personal communication). This observation could shed some 

light on the relative activity of other heterocyclic replacements for the 

isoxazole ring. 

A. HRV-14 Model Development 

The extensive data generated from x-ray studies with HRV-14 permitted 

the development of a model that could define the properties required of this 

class of compounds for antiviral activity [37]. This model was dependent on 

the orientation and x-ray conformational data for compounds bound to 

the viral pocket. Some assumptions were made based on earlier results and 

on rules generated for predicting compound orientation. For example, it 


Figure 12 Hydrogen bonding network involved in the binding of WIN54954 

analogues to HRV-14. 

was assumed that all the compounds included in the study that had not 

been examined by x-ray crystallography behaved in a predictable manner. 

Compounds were divided into two groups of seven compounds each. One 

group whose conformations were known (Fig. 13) demonstrated various 

levels of activity against the virus. The second group consisted of inactive 

compounds with related structures (Fig. 14). In the absence of conformational 

data for this group, one of the active compounds was used as a template 

for these compounds. A SYBYL (version 5.0) database was created. 

All the structures were overlaid in the position found in the binding site 

(Fig. 15). Volume maps were then calculated for the Boolean ‘‘union’’ of all 

active and inactive compounds, which were then overlaid, and the excess 

volume occupied by the inactive compounds, in comparison to the active 

compound (Boolean minus), was calculated (Fig. 16). A similar procedure 

was followed for the excess volume for the actives (Boolean plus). These 

combined results revealed that inactive compounds displayed excessive 

bulk around the phenyl ring. Although some bulk is desirable in this 

area to enhance hydrophobic interactions, excessive bulk, which leads to 


Figure 13 Compounds active against HRV-14, which were used in the development 

of the volume map model. 

steric interactions, leads also to inactivity [38]. Conversely, space occupancy 

in the pore area of the binding site was found to contribute to good 

biological activity. 

To refine this model qualitatively, the binding of several of these 

compounds was subjected to a CoMFA (Comparative Molecular Field 

Analysis) [39]. This program examines electrostatic and steric parameters 


Figure 14 Compounds inactive against HRV-14. 

and through a partial-least-squares analysis determines the correlation of 

these effects with biological activity. Either rigid structures or fixed 

conformations are required to carry out the analysis. Eight compounds, 

which had been used in the volume map study and whose binding 

conformations were known, were employed. These compounds also had 

a reasonable spread of activity against HRV-14. In addition, they offered 

some degree of structural diversity. Van der Waals radii for atoms were 

taken from a standard Tripos force field. Charges were calculated by the 

AMI method by single-point calculations on the receptor-bound conformation 

of the drug molecule. Point charges on the hydrogen atoms were 

not collapsed onto the atom to which they were bound but were left on 

the hydrogen atoms. Log p values were calculated using the MedChem 

software package (version 3.54). All these parameters, in addition to the 

CoMFA field values designated by *, were used in the quantitative 

structure–activity (QSAR) analysis (Fig. 17). 


Figure 15 Overlay of active and inactive compounds. 

Structural alignments were obtained from x-ray crystallographic 

analysis. The backbone residues within 20 A ˚ of any atom of the compound 

were included in this study. This effectively created a cube 20 A ˚ on 

a side, which was divided into grid points 1 A ˚ apart. A hydrogen atom 

and a proton as a probe were used to sample each grid point for both 

electrostatic and steric effects. The data were tabulated and cross-validated 

along with the physical parameters by means of a partial-least-squares 

method, with the following results: good correlation of MIC with CoMFA 

data and good predicative capabilities in the case of steric properties 

(Fig. 18). No meaningful correlation was seen with electrostatic parameters, 

either taken in combination with steric factors or evaluated alone. 

A regression analysis using all the values shown in Figure 17 revealed no 

contribution of any parameters, other than the CoMFA field, to the activity 

of these compounds. 

In addition to the QSAR data, which resulted from this program, 

three-dimensional contour maps were generated for both steric and elec- 


Figure 16 Overlay of volume maps of active compounds and inactive 

compounds. 

trostatic fields. Cutoffs were used to contour together points where the 

correlations were highest and positive and those that were highest and 

negative. Although the shapes of the maps coincide with the shape of the 

pocket, the structure of the macromolecules was not part of the calculations. 

The visual results displayed by the contour maps qualitatively 

agree with the QSAR results; that is, there is no significant correlation 

between electrostatics and biological activity (Fig. 19), despite a strong 

correlation between the steric fields and activity, as predicted. Although 

a moderate positive effect was seen in the vicinity of the aromatic ring, 

in general, this model predicts that excessive bulk in this area negatively 

correlates with biological activity. These results are in agreement with the 

conclusions empirically generated from the volume map study and also 

confirm the lack of electrostatics involved in the phenyl–phenyl stacking 

interactions, which had been observed earlier. 


Figure 17 CoMFA coordinates. 

B. Model Development Based on Small-Molecule, 

Low-Energy Conformations 

Thus far, the model development discussed has been one based on x-ray 

conformations. Considering that there are over 100 serotypes, the discovery 

of broad spectrum antirhinovirus agents would require considerably 

more three-dimensional virus structures. We have investigated the possibility 

of simply using energy-minimized small-molecule conformations 

exclusive of the virus structures [40]. The compounds in question were 

constructed in SYBYL and minimized by means of Maximin. By using a 

template for spatial referencing (Fig. 20) that represented one of the more 

potent compounds against HRV-14, it was possible to employ the program 

Superimpose (SYBYL version 5.0) to algorithmically overlay the molecules 

based not on conformational similarity but rather on shape. Volume 

maps were then constructed as already described. Figure 21 compares 

the volume maps created by this method with those from x-ray structures 

in HRV-14. The difference maps clearly show that within certain limits, 

increasing chain length increases activity. Although there is a space-filling 

requirement for activity, exceeding the appropriate distribution or extent 

of bulk results in inactive compounds. These findings essentially duplicate 

those obtained from the preceding method. 


Figure 18 Regression equation obtained from partial-least-squares data crossvalidated 

in Figure 17. 

This procedure was repeated for HRV-1A. Duplicate maps were 

generated by means of both procedures and clearly show that shorter 

molecules, as measured from the phenoxy to isoxazole moieties, are more 

active. Molecules with the correct degree and placement of bulk in the 

middle of the volume are also more active. These encouraging results 

suggest that this method can be applied to other serotypes without giving 

consideration to their three-dimensional structures. 

C. Application of Model Development to Drug Design 

The results of the model development for HRV-14 and HRV-1A demonstrated 

that the problem of drug design is complicated by the difference 

in the dimensions of the binding sites, at least in the case of these two 

serotypes. One solution to this problem is to prepare a compound that 


Figure 19 Three-dimensional contour map generated from CoMFA analysis of 

electrostatic fields showing correlation with antiviral activity. Red indicates a 

strong correlation. The grid map is a result of an analysis using a probe atom (charge 

0). All charges on the molecules were calculated using the AM1 Hamiltonian 

without geometry optimization. 

has some degree of flexibility and would be accommodated by the binding 

sites in both serotypes. We chose to examine the homologous series 

shown in Figure 22. The three-carbon bridge structure demonstrated good 

activity against HRV-1A but poor activity against HRV-14. As the flexible 

side chain is increased, a concomitant improvement in activity is seen 

against HRV-14, with optimum activity observed against both serotypes 

with the three-carbon side chain. This avenue was pursued because further 

testing of the three-carbon chained analogues against 100 rhinovirus 

serotypes indicated that these compounds exhibited a broader spectrum 

of activity, suggesting that perhaps HRV-14 is not representative of the 

majority of serotypes. 


Figure 20 Minimized structure serving as a template for spatial referencing in the 

model development using energy-minimized small molecules. 

D. The Development of a Clinical Candidate 

Biological activity is not the only criterion required for drug development, 

as anyone who has been involved in this area is aware. Potency, toxicity, 

bioavailability, metabolic stability, and plasma half-life are only a few of 

the critical issues that must be addressed. Although satisfactory potency 

and spectrum activity had been achieved with WIN54954, which has been 

clinically evaluated, this compound lacked metabolic stability and consequently 

displayed a short half-life. 

It became clear that the oxazoline ring was metabolically unstable 

and was responsible for the generation of crystalurea with disoxaril and for 

a drug-induced rash with WIN54954, accompanied by a short plasma halflife. 

Consequently, a replacement for the oxazoline ring was sought, which 

would be metabolically stable and would demonstrate satisfactory bioavailability. 

After examining several heterocyclic replacements, the 4- 


Figure 21 Comparison of volume map for HRV-14 generated from x-ray data 

(left) and small-molecule energy-minimized structures (right). 

methyltetrazole analogue with a three-carbon linker (Fig. 23) appeared to 

provide good chemical stability and improved biological activity in 

comparison to WIN54954 [41]. However, when this compound was 

administerd to dogs, hepatotoxicity was observed which was attributed 

to metabolic instability. Further modifications resulted in the synthesis of 

the 5-methyl 1,2,4-oxadiazole analogue, which was selected as a possible 

Figure 22 Homologous series of compounds. 


Figure 23 Structure of the 4-methyltetrazole analogues. 

development candidate based on potency and spectrum of activity [42]. To 

address metabolic stability, however, a monkey liver microsomal assay 

was established by means of which the half-life, the extent of metabolism, 

and the nature of the metabolic products could be determined [43]. Initially, 

WIN54954 was incubated at 37jC with a liver microsomal mixture 

for 30 min and the incubate was extracted with hexane. The extracts were 

analyzed by high performance liquid chromatography (HPLC), which 

revealed 18 metabolic products. When the oxadiazole analogue was subjected 

to the same conditions, two major peaks, metabolites A and B, were 

observed by HPLC (Fig. 24), in addition to six minor ones. The rate of 

metabolism was similar to that of WIN54954, however, with a half-life of 

27 vs 20 min. 


Figure 24 HPLC spectrum resulting from the incubation of the oxadiazole with 

monkey liver microsomes. 

The question at this point was whether modifications could be made 

to the oxadiazole molecule to enhance metabolic stability and achieve 

comparable activity. This approach required knowledge of the site of 

metabolism and the nature of the metabolic products. This information 

was obtained from ion mass spectrometry. The identity of these products 

was determined by comparing the fragmentation pattern of metabolites A 

and B with the parent compound and the corresponding daughter ions 

(Fig. 25). 

Analysis of the metabolic products indicated that hydroxylation 

occurred to a greater extent (30%) on the methyl group attached to the 

isoxazole ring than to the methyl group on the oxadiazole ring (10%). The 

methyl group in this postion was replaced with a trifluormethyl group to 

prevent hydroxylation. The result of the incubation of this compound 

indicated that although this position was protected, three metabolic 

products were produced; in addition, the half-life was not substantially 

different from the parent compound. 

A similar replacement on the oxadiazole ring (Fig. 26) not only prevented 

metabolism at this position but also protected the entire mole- 


Figure 25 Biotransformation of WIN 61893 and WIN 64172. 

Figure 26 Metabolism of the trifluoromethylisoxazole analogue. 


Figure 27 Structure of second properties pleconaril. 

cule, resulting in two minor metabolites, and substantially increased the 

half-life. In addition, pleconaril has exhibited a broad spectrum of antipicornavirus 

activity and has shown good bioavailability (Fig. 27) and is 

undergoing clinical trials for upper respiratory rhinovirus infections. 

V. CONCLUSIONS 

x-Ray crystallography has added a new dimension to antirhinovirus drug 

design. It has enabled us to examine the molecular interactions within 

the compound binding site and to better understand the mechanism of 

binding. We have been able to devise a model based on x-ray crystallography 

that qualitatively describes properties of molecules that are beneficial 

for antirhinovirus activity. Also, by comparison to a volume map based 

on x-ray conformations, we have developed a comparable model based on 

small-molecule energy-minimized structures exclusive of x-ray data. 

Finally, we have been able to apply our results to the synthesis of compounds 

active against both HRV-1A and HRV-14. Aside from the design 

aspects, we have dealt with the more practical considerations such as 

metabolic stability and bioavailability, which have led to a clinical candidate. 

Now certain unanswered mechanistic questions can be addressed. 

How does the drug enter the binding site? Is there a recognition site, which 

may explain some anomalous results that remain a mystery? Hopefully, 


future work in this regard will eventually lead to an understanding of the 

binding process and its relationship to biological activity. 

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11 

Profiles of Prototype Antiviral Agents 

Interfering with the Initial Stages 

of HIV Infection 

Erik De Clercq 

Rega Institute for Medical Research, Katholieke Universiteit Leuven, 

Leuven, Belgium 


The initial stages of the human immunodeficiency virus (HIV) infection 

could be defined as the steps of the viral growth cycle that precede the 

integration of the proviral DNA into the host cell genome. These stages 

occur during the acute phase of the HIV infection, that is, when the virus 

has invaded new cells. Once the proviral DNA has been integrated into 

the host genome, the host cell and all its progeny cells can be considered to 

be persistently or chronically infected. Expression of the integrated viral 

genome will follow the classical flow of gene expression: that is, transcription, 

translation, and post-translational modifications under the concerted 

regulatory action of both cellular and viral factors. 

This chapter reviews prototypes of single chemical entities that 

interfere with the initial stages of the acute HIV infection, at steps that 

are predominantly, if not solely, determined by specific viral proteins. 

Thus, the compounds interacting with these steps in the HIV replicative 

cycle may be expected to display a reasonably high specificity in their 

mode of action. The targets that could be envisaged for such chemotherapeutic 

attack are the following: (1) virus adsorption, involving the 


viral envelope glycoprotein gp120, (2) virus–cell fusion, involving both 

viral glycoproteins gp120 and gp41, (3) viral uncoating, involving the viral 

capsid proteins, (4) the substrate (dNTP) binding site of the viral reverse 

transcriptase, and (5) an allosteric non–substrate binding site of the HIV-1 

reverse transcriptase. HIV inhibitors interacting with these targets have 

been the subject of some earlier reviews [1–4]. The chemokine receptors 

CXCR4 and CCR5 used as coreceptors by X4 and R5 HIV-1 strains are 

not discussed here; for reviews on inhibition of HIV infection by these receptor 

antagonists, see Refs. 5 and 6. 

The compounds are highlighted from the following viewpoints: anti- 

HIV potency and selectivity, mechanism of action, antiviral activity spectrum, 

clinical or therapeutic potential, and risk of resistance development. 

II. VIRUS ADSORPTION INHIBITORS: 

POLYANIONIC SUBSTANCES 

Various polyanionic substances (viz., polysulfates, polysulfonates, polycarboxylates, 

and polyoxometalates) have been reported to block HIV 

replication; for a review on the polysulfates, see Ref. 7. These substances 

inhibit HIV-induced cytopathicity at a concentration of 0.1 to 1 Ag/mL, 

while not being toxic to the host cells at concentrations up to 2 or 5 mg/mL, 

thus achieving selectivity indexes of approximately 10,000 [7]. The target of 

interaction for the polysulfates would be the V3 loop of the viral gp120 

glycoprotein [8–10]. This loop contains a highly basic region with which 

the polyanionic substances could interact electrostatically. Thus, polyanions 

such as dextran sulfate may be assumed to block virus adsorption by 

shielding the viral envelope glycoproteins [8]. Alternatively or additionally, 

polyanionic substances may also interact with the cellular CD4 receptor 

[11], thus preventing the viral envelope gp120 from anchoring to 

the outer cell membrane. 

Depending on their molecular weight, the nature of their anionic 

groups, and the density/distribution of their negative charges, the polyanionic 

substances exhibit an activity spectrum that extends to several 

enveloped viruses other than HIV: among the retroviruses, SIV (simian 

immunodeficiency virus); among the herpesviruses, HSV (herpes simplex 

virus) and CMV (cytomegalovirus); among the orthomyxoviruses, influenza 

A; among the paramyxoviruses, RSV (respiratory syncytial virus); 

and toga-, flavi-, arena-, bunya-, and rhabdoviruses. Among the different 

HIV strains, rather striking differences have been noted with regard to 


susceptibility to polyanionic substances (e.g., dextran sulfate) [12], and this 

differential susceptibility may be related to differences in the composition 

of the viral glycoprotein portions with which the compounds interact. 

Because of their broad activity spectrum, encompassing various 

enveloped viruses, polyanionic substances may be of practical utility in 

the prophylaxis and/or therapy of a number of important virus (e.g., HIV, 

HSV, CMV, RSV, influenza A) infections. Yet, there is little, if any, 

evidence for the in vivo efficacy of these compounds following either 

parenteral or topical administration. Polyanions, and dextran sulfate in 

particular, are poorly absorbed upon oral administration [13], and, in 

addition, sulfated polysaccharides are notorious for their anticoagulant 

activity. However, these problems can be overcome by the appropriate 

chemical modifications (Fig. 1). Thus, h-cyclodextrin sulfate becomes 

orally bioavailable following substitution of benzyl groups at either C-2 

or C-6 of the sugar residues mCDS71 [14] and mCDS11 [15], respectively, 

and heparin loses anticoagulant activity when acylated at the C-3 position 

of the sugar rings [16]. These favorable features (oral bioavailability, loss of 

anticoagulant activity) were obtained without impairing the anti-HIV 

activity of the products (mCDS71, mCDS11, or O-acylated heparin). 

The polyanionic substances may be expected to yield their greatest 

promise when put in contact with the virus under the conditions that 

Figure 1 Modified sulfated polysaccharides: (A) O-acylated heparin (m = 2, 4, 

6, ...) and (B) mCDS71 (a modified h-cyclodextrin sulfate). 


mimic as closely as possible the in vitro situation, where these compounds 

have proved so clearly effective. Further studies should address such issues 

as delivery forms, route(s) of administration, and time of treatment (with 

respect to the virus infection), before an appropriate candidate compound 

is submitted to clinical trials. Polyanionic substances are not known to 

lead to resistance development, although, as mentioned, different HIV 

strains may differ markedly ab initio in their susceptibility to this class of 

compounds. 

III. VIRUS–CELL FUSION INHIBITORS: LECTINS, 

ALBUMINS, AND TRITERPENE DERIVATIVES 

Because of their interference with the interaction between the viral 

envelope gp120 glycoprotein and the cellular CD4 receptor, polyanionic 

substances not only inhibit virus adsorption to the cells but also block 

syncytium (giant cell) formation between the HIV-infected (gp120 + ) cells 

and uninfected (CD4 + ) cells. Since syncytium formation results in a 

selective destruction of the CD4 + cells, this syncytium formation may 

play an important role in the pathogenesis of AIDS (a hallmark of which is 

a progressive decline of the CD4 + cells). 

There are a number of compounds known to block syncytium 

formation without (markedly) affecting virus binding to the cells. These 

compounds may therefore be assumed to directly interfere with the virus– 

cell fusion process, that is, fusion between the viral envelope and the outer 

cell membrane. The compounds that have been postulated to inhibit 

virus–cell fusion include the following: mannose-specific lectins (i.e., from 

Listeria ovata, Hippeastrum hybrid, Cymbidium hybrid, and Epipactis 

helleborine) andN-acetylglucosamine-specific plant lectins (i.e., from 

Urtica dioica) [17,18]; a derivative from polyphemusin, a peptide that is 

highly abundant in hemocyte debris of the horsehoe crab Limulus polyphemus 

[19]; succinylated human serum albumin, Suc-HSA [20], and 

aconitylated human serum albumin, Aco-HSA [21] (Fig. 2); and triterpene 

(i.e., betulinic acid) derivatives (Fig. 3) [22]. Research into the efficacy 

of a number of natural products that have been described as anti-HIV 

agents is reviewed in Ref. 23. 

Despite their widely varying origin and structure, these different 

classes of compounds seem to be targeted at the virus–cell fusion process, 

although the exact mechanism by which the compounds inhibit fusion, 


Figure 2 Modified human serum albumins from HSA: succinylated human 

serum albumin (Suc-HSA) and aconitylated human serum albumin (Aco-HSA). 

and the target amino acid sequences (at gp120 and/or gp41) with which 

they putatively interact, remain to be elucidated. Most of the compounds 

inhibit HIV replication at concentrations of 0.1 to 1 Ag/mL and some 

(Aco-HSA and betulinic acid) are even effective within the concentration 

range of 0.01 to 0.1 Ag/mL [21,22]. For the plant lectins [17,18] and modified 

serum albumins [20,21], the inhibitory effects on HIV replication correlated 

closely with their inhibitory effects on syncytium formation, which 

corroborates the hypothesis that their anti-HIV activity is due to inhibition 

of virus–cell fusion. 

Whereas the plant lectins are inhibitory to HIV-1, HIV-2, and a 

number of other (enveloped) viruses (viz., CMV, RSV, influenza A), at 


Figure 3 Triterpene derivative: betulinic acid RPR103611: NV-{N-[3h-hydroxylup-20(29-ene-28-oyl]-8-aminooctanoyl}-L-statine. 

concentrations well below the cytotoxicity threshold, the succinylated and 

aconitylated albumins inhibit only HIV, HIV-2 being (much) less susceptible 

to these compounds than HIV-1 [20,21]. Betulinic acid is even more 

restricted in its antiviral activity spectrum in that it is active only against 

HIV-1, and not even all HIV-1 strains [22]: specifically, betulinic acid is 

not active against the NDK strain of HIV-1. This must point to a highly 

specific molecular site for the interaction of betulinic acid [22] and should 

help in deciphering the target amino acid sequences (at gp120 or gp41) for 

this compound. 

The clinical potential of the fusion inhibitors in the therapy and/or 

prophylaxis of HIV infections remains a subject for further study. Since 

these compounds directly interfere with syncytium formation, they should 

be able to block HIV infections generated by both free virus particles and 

HIV-infected cells. It is not known how readily the virus may become 

resistant to this class of compounds. For betulinic acid, it has been 

ascertained that some HIV-1 strains (e.g., NDK) may be resistant ab initio. 

IV. VIRUS UNCOATING INHIBITORS: BICYCLAMS 

Bicyclams (Fig. 4) consist of two cyclam (1,4,8,11-tetraazacyclotetradecane) 

units tethered via an aliphatic (i.e., propylene, as in JM2763) or 

aromatic bridge [i.e., phenylenebis(methylene), as in JM3100]. While the 

bicyclam JM2763 inhibits HIV-1 and HIV-2 replication at a concentration 

of 0.1 to 1 Ag/mL [24], the bicyclam JM3100 does so at a hundredfold 

lower concentration, that is, at a concentration that is more than 


Figure 4 Bicyclam derivatives (A) JM2763 and (B) JM3100, each consisting of 

two cyclam (1,4,8,11-tetraazacyclotetradecane) moieties tethered via a propylene 

(JM2763) or phenylenebis(methylene) bridge (JM3100). 

100,000-fold lower than the cytotoxic concentration [25]. In primary T4 

lymphocytes and monocytes (macrophages), JM3100 inhibits HIV-1 replication 

at concentrations lower than 1 ng/mL [25]. 

The bicyclams represent the only retrovirus inhibitors that have been 

postulated to interfere with the viral uncoating process. This assumption 

has been based on ‘‘time of addition’’ experiments where the compounds 

(JM2763 and JM3100) were found to act at a stage following virus adsorption 

but preceding reverse transcription; and, since the compounds 

did not prove inhibitory to syncytium formation (JM2763) [or were 

inhibitory only at a concentration substantially higher than that required 

for inhibition of HIV replication (JM3100)], their target of action could be 

tentatively identified as a viral uncoating event. This hypothesis was then 

corroborated by ‘‘uncoating’’ experiments in which the viral RNA, 

recovered from HIV-infected cells that had been exposed to the compounds, 

was monitored for sensitivity to ribonuclease A: the viral RNA 

was protected against degradation by RNase A, as could be anticipated if 

the uncoating (i.e., dissociation of the capsid proteins from the viral RNA) 

had been impeded [24,25]. 

Current investigations are attempting to determine with which viral 

(capsid) proteins, and which amino acid residues of their target proteins, 

the bicyclam interact. The antiviral activity spectrum of the bicyclams is 

clearly different from that of other anti-HIV agents in that the bicyclams 

are equally effective against HIV-1 and HIV-2 but less effective or not 

active against SIV (in human T lymphocytes). Given its high selectivity 

index ( >100,000) in vitro, the bicyclam JM3100 offers great potential for 

the treatment of HIV-1 and HIV-2 infections in humans. Although the 

bicyclams may under some conditions select out drug-resistant variants 

from clinical HIV-1 (i.e., HE) strains, it has otherwise proved difficult, or 


even impossible, to generate resistance to the compounds (i.e., JM3100) 

after repeated passages in cell culture [25]. 

V. REVERSE TRANSCRIPTASE INHIBITORS 

INTERACTING WITH THE SUBSTRATE 

BINDING SITE 

A. Dideoxynucleoside Analogs 

In addition to the three anti-HIV agents [AZT (zidovudine), DDI (didanosine) 

and DDC (zalcitabine)] that have been formally approved by the 

U.S. Food and Drug Administration for the treatment of HIV infections, 

several other 2V,3V-dideoxynucleoside (ddN) analogues (Fig. 5), including 

3V-fluoro-2V,3V-dideoxy-5-chlorouridine (FddClUrd) and 2V,3V-didehydro- 

Figure 5 Dideoxynucleoside (ddN) analogues: 2V,3V-dideoxycytidine (DDC), 3Vazido-2V,3V-dideoxythymidine 

(AZT), 3V-fluoro-2V,3V-dideoxythymidine (FLT), 

2V,3V-didehydro-2V,3V-dideoxythymidine (D4T), 3V-thia-2V,3V-dideoxycytidine 

(3TC), 3V-thia-2V,3V-dideoxy-5-fluorocytidine (FTC), and 2V,3V-dideoxy-L-cytidine 

(L-DDC). 


2V,3V-dideoxythymidine [D4T (stavudine)], have been reported to inhibit 

HIV replication (for review, see Refs. [26–28]). In particular, FddClUrd 

appears to be an attractive candidate for further development, since it is 

much less toxic to the host cells than AZT and most other ddN analogues 

[29,30]. Also ranking among the most promising ddN analogues are 3Vthia-2V,3V-dideoxycytidine 

[3TC (lamivudine)] and 3V-thia-2V,3V-dideoxy- 

5-fluorocytidine (FTC), which are actually more active in their (–)-h- or 

L-isomeric form than in the (+)-h- orD-isomeric form [31,32]. 

All ddN analogues, including 3TC and FTC, act in a similar fashion; 

that is, following intracellular phosphorylation to their 5V-triphosphate 

form, they serve as competitive inhibitors/alternate substrates of the reverse 

transcriptase (RT) reaction, thus leading to chain termination, as 

has been clearly demonstrated with AZT [33]. The anti-HIV activity of 

ddN analogues is critically dependent on their intracellular phosphorylation, 

the first phosphorylation step being the most crucial. For some 

compounds (viz., 2V,3V-dideoxyuridine) and in some cells (viz., monocytes/macrophages), 

the nucleoside kinase activity of the cells may be 

inadequate to satisfactorily accomplish the first phosphorylation step; and 

thus prodrugs, including aryl methoxyglycinyl derivatives [34] and bis[S- 

(2-hydroxyethylsulfidyl)-2-thioethyl] esters [35] have been designed that 

deliver the 5V-monophosphate form intracellularly, bypassing the first 

phosphorylation step. 

The antiviral activity spectrum of the ddN analogues should, in 

principle, extend to all retroviruses as well as hepadnaviruses [i.e., hepatitis 

B virus (HBV)], since HBV, like retroviruses, replicates through an 

RNA template-driven RT process. Indeed, various ddN analogues (particularly, 

the L-enantiomeric forms 3TC, FTC, and L-DDC) have been 

shown to inhibit HBV replication [36–38]. Consequently, 3TC is, at present, 

pursued as a potential drug candidate for the treatment of both HIV 

and HBV infections. 

Prolonged AZT therapy of HIV-infected individuals leads to a reduction 

of virus sensitivity to the drug [39]. This reduced sensitivity, generally 

termed ‘‘resistance,’’ appears to be based on the following mutations 

in the HIV-1 RT [40,41]: 41 Met ! Leu, 67 Asp ! Asn, 70 Lys ! Arg, 

215 Thr ! Phe/Tyr, and 219 Lys ! Gln. Of these mutations, the 

215 Thr ! Tyr mutation has been the most frequently detected among 

AZT-resistant HIV isolates from patients under prolonged AZT therapy 

[42]. The 74 Leu ! Val mutation is responsible for resistance to DDI [43], 

and the 184 Met ! Val mutation confers resistance to 3TC, FTC, DDC, 

and DDI [44–46]. 


The mutations at position 74 (Leu ! Val) and 184 (Met ! Val) of 

the HIV-1 RT do not lead to cross-resistance to AZT. Nor would the 215 

Thr ! Tyr mutation lead to cross-resistance to 3TC, FTC, DDC, or DDI. 

In fact, the mutations at positions 74 and 215 seem to counteract each 

other, and so do the mutations at positions 184 and 215. Based on this 

‘‘mutually counteracting mutation’’ principle [47], drug combinations 

could be envisaged that, if combined, might counteract emergence of resistance 

to one another: namely, combinations of AZT with either DDI, 

3TC, FTC, or DDC. As will be explained further, these two-drug combinations 

may be extended to three-drug or four-drug combinations, following 

the addition of one or more of the HIV-1-specific nonnucleoside RT 

inhibitors (NNRTIs). 

B. Acyclic Nucleoside Phosphonates 

Acyclic nucleoside phosphonates (ANPs) (Fig. 6) may be regarded as 

analogous to the ddN monophosphates, thus allowing us to circumvent the 

first phosphorylation step required for the intracellular activation of the 

compounds. After they have been taken up as such by the cells, the acyclic 

nucleoside phosphonates (PMEA, PMEDAP, PMPA, PMPDAP, 

FPMPA, and FPMPDAP) are converted intracellularly to their respective 

diphosphate form (PMEApp, PMEDAPpp, PMPApp, PMPDAPpp, 

FPMPApp, and FPMPDAPpp) and, in such form they interact as competitive 

inhibitors, alternate substrates, or chain terminators with the reverse 

transcriptase [48–50]. 

PMEA and its congeners are more effective in vivo than could be 

predicted from their in vitro potency. While less potent as an antiretrovirus 

agent than AZT in vitro, PMEA proved clearly superior to AZT 

when the two drugs were compared for their effectiveness in vivo, in mice 

infected with murine Moloney sarcoma virus [51,52]. PMEA was also 

shown to be effective against various other retrovirus infections, including 

Friend leukemia virus (FLV), Rauscher leukemia virus (RLV), and LP- 

BM5 (murine AIDS) virus infection in mice, feline leukemia virus (FeLV) 

or feline immunodeficiency virus (FIV) infection in cats, and SIV infection 

in macaque (rhesus) monkeys (for review, see Ref. 53). In the latter model 

[54], again PMEA proved far superior to AZT in suppressing several 

parameters of the disease. 

The antiviral activity spectrum of PMEA, PMEDAP, and their 

congeners is not confined to retroviruses but also extends to hepadnaviruses 

(e.g., HBV). PMEA has proved effective against duck HBV infec- 


Figure 6 Acyclic nucleoside phosphonates (ANPs): 9-(2-phosphonylmethoxyethyl)-adenine 

(PMEA) and -2,6-diaminopurine (PMEDAP), (R)-9-(2-phosphonylmethoxypropyl)-adenine 

(PMPA) and -2,6-diaminopurine (PMPDAP), (S)-9-(3fluoro-2-phosphonylmethoxypropyl)-adenine 

(FPMPA) and -2,6-diaminopurine 

(FPMPDAP), and the bis(pivaloyloxymethyl) ester of PMEA [Bis(pom)-PMEA]. 

tion in both duck hepatocytes and Pekin ducks [55]. For PMEA and 

PMEDAP, but not for PMPA, PMPDAP, FPMPA, or FPMPDAP, the 

activity spectrum also extends to herpesviruses (e.g., HSV, CMV). This 

would make PMEA and PMEDAP particularly attractive as therapeutic 

modalities in AIDS patients, since they might be useful not only for the 

treatment of the underlying HIV infection but also for the therapy/ 

prophylaxis of the intercurrent HSV or CMV infections. 


Another attractive feature of PMEA, and, in fact, all ANPs, is 

prolonged antiviral action, lasting for several days, or even one week or 

longer, after a single-dose administration. This long-lasting antiviral 

action may be related to the long half-life of the active metabolites (e.g., 

PMEApp) within the cells and may permit infrequent (e.g., weekly) dosing 

of the ANPs in the prophylaxis and/or therapy of (retro)virus infections. 

Little is known on how readily or rapidly retro- or herpesviruses may 

develop resistance to the ANPs. In the in vitro and in vivo experiments 

done so far with PMEA, PMEDAP, or any of the other ANPs, resistance 

development did not seem to occur, but further studies are needed to 

address this issue. 

Since the ANPs are only slowly taken up by the cells and poorly 

absorbed following oral administration, some efforts have been directed 

toward the development of prodrugs (esters) that would be better taken up 

by the cells. These efforts have yielded the bispivaloyloxymethyl [bis(pom)] 

derivative of PMEA (Fig. 6) [56]. Bis(pom)-PMEA shows a cellular uptake 

increased more than a hundredfold, as well as fivefold better oral 

bioavailability than the parent compound [57]. Both PMEA (given intravenously) 

and bis(pom)-PMEA (given perorally) are now in clinical trials 

in patients with AIDS. 

VI. REVERSE TRANSCRIPTASE INHIBITORS 

INTERACTING WITH A NONSUBSTRATE 

BINDING SITE: NON-NUCLEOSIDE REVERSE 

TRANSCRIPTASE INHIBITORS 

The identification of the HIV-1-specific non-nucleoside reverse transcriptase 

inhibitors (NNRTIs) as a separate class of HIV inhibitors was 

heralded by the discovery of the tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one 

and -thione (TIBO) derivatives (Fig. 7) [58,59] and 

1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivatives 

(Fig. 8) [60,61]. The first TIBO derivatives (R82150, R82913) were the 

first NNRTIs [58] postulated to act as inhibitors of HIV-1 RT [59]. For the 

HEPT derivatives it became evident that they also interact specifically 

with HIV-1 RT after a number of derivatives (i.e., E-EPU, E-EBU, and 

E-EBU-dM) had been synthesized that were more active than HEPT 

itself [62,63]. Following HEPT and TIBO, several other compounds, i.e., 

nevirapine, pyridinone, and bis(heteroaryl)piperazine (BHAP), were 


Figure 7 Tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H )-one (TIBO) derivatives 

(A) R82913 and (B) R86183 (with a chlorine substituted in the 9- or 8position, 

respectively). 

Figure 8 (A) 1-(2-Hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT). (B) 5- 

Isopropyl-1-(ethoxymethyl)-6-benzyluracil (I-EBU, MKC-442). 


described as HIV-1-specific RT inhibitors (for a review on the HIV-1specific 

RT inhibitors, see Refs. 28 and 64). 

The HEPT and TIBO derivatives were discovered as the result of a 

systematic evaluation for anti-HIV activity in cell culture. They were later 

found to achieve their anti-HIV-1 activity through an interaction with the 

HIV-1 RT. In contrast, nevirapine, pyridinone, and BHAP emerged from 

a screening program for HIV-1 RT inhibitors. The anti-HIV-1 activity 

of these compounds was subsequently confirmed in cell culture. Like the 

HEPT and TIBO derivatives, the 2V,5V-bis-O-(tert-butyldimethylsilyl)-3Vspiro-5VV-(4VV-amino-1VV,2VV-oxathiole-2VV,2VV-dioxide)-pyrimidine 

(TSAO) derivatives 

(Fig. 9) [65,66] and a-anilinophenylacetamides (a-APA) (Fig. 10) 

[67] were discovered through the evaluation of their anti-HIV activity in 

cell culture. Subsequently, they were found to act as specific inhibitors of 

HIV-1 RT. 

Yet other compounds have been found to inhibit HIV-1 replication 

through a specific interaction with HIV-1 RT (i.e., quinoxaline S-2720 [68], 

5-chloro-3-(phenylsulfonyl)indole-2-carboxamide [69], dihydrothiazoloisoindolones 

[70] and a number of natural substances (e.g., calanolide A 

and inophyllums, from the tropical rain forest trees Calophyllum lanigerum 

and Calophyllum inophyllum, respectively) [71,72]. All these and yet other 

compounds could be considered to be NNRTIs. The most potent among 

the NNRTIs, some of the HEPT derivatives (E-EBU-dM) [63] and a- 

Figure 9 2V,5V-Bis-O-(tert-butyldimethylsilyl)-3V-spiro-5W-(4W-amino-1W,2W-oxathiole-2W,2W-dioxide)pyrimidine 

(TSAO) derivatives TSAO-T, TSAO-m 3 T, and 

TSAO-e 3 T. 


Figure 10 a-Anilinophenylacetamide (a-APA) derivatives (A) R18893, (B) 

R88703, and (C) R89439. 

APA derivatives (R89439) [67], inhibit HIV-1 replication at a concentration 

of approximately 1 ng/mL, that is, 100,000-fold below the cytotoxicity 

threshold. 

While the ddNs and ANPs must be converted intracellularly to 

their 5V-triphosphates (ddNTPs) or diphosphate derivatives before they 

can interact as competitive inhibitors/alternate substrates with regard to 

the natural substrates (dNTPs), the NNRTIs do not need any metabolic 

conversion to interact, noncompetitively with respect to the dNTPs, at 

an allosteric, non–substrate binding site of the HIV-1 RT. Through the 

analysis of NNRTI-resistant mutants, combined with site-directed mutagenesis 

studies, it has become increasingly clear which amino acid 

residues are involved in the interaction of the NNRTIs with HIV-1 RT, 

and, since the conformation of the HIV-1 RT has been resolved at 3.0 

A ˚ resolution [73], it is now possible to visualize the binding site of the 

NNRTIs [74]. 

The antiviral activity spectrum of the NNRTIs is limited to HIV-1, 

probably because only HIV-1 RT contains a pocket site at which the 


NNRTIs may bind. The high specificity displayed by the NNRTIs in 

their binding to HIV-1 RT signals that it should, a priori, be relatively 

easy for the enzyme (and the virus) to escape the inhibitory effects of the 

NNRTIs through mutations of the amino acid residues that either are 

directly involved in the binding of the NNRTIs or contribute to the configuration 

of the pocket that is ideal for NNRTI binding. 

From pilot studies carried out in the clinic with the NNRTIs TIBO 

R82913 [75] and pyridinone L-697,661 [76], it appears that the compounds 

are well tolerated and do not cause toxic side effects. Most of the HIV-1 

isolates obtained from the patients treated with TIBO R82913 appeared to 

be as sensitive to the compound as wild-type virus; only two HIV-1 

variants were isolated, showing a sensitivity that was reduced 20-fold or 

more than 100-fold, the latter being caused by a mutation (Tyr ! Leu) at 

position 188 of the RT [77]. In fact, the latter mutation was lost upon 

passaging the virus in vitro in cord blood lymphocytes. Following treatment 

of the patients with pyridinone L-697,661, drug-resistant HIV-1 

variants appeared that contained mutations at the RT positions 103 (Lys 

! Asn) and 181 (Tyr ! Cys) [76]. 

HIV-1 resistance to NNRTIs rapidly arises following passage of the 

virus in cell culture in the presence of the compounds. The 181 Tyr ! Cys 

mutation is most commonly seen, and it leads to resistance, or at least to 

reduced sensitivity, to most of the NNRTIs (i.e., TIBO, HEPT, nevirapine, 

pyridinone, BHAP, TSAO, a-APA) [78–84]. The 188 Tyr ! His mutation 

is associated with resistance to TIBO [85], but not nevirapine [82]. 

The 103 Lys ! Asn mutation is associated mainly with resistance to TIBO 

and pyridinone [78,85]. The 100 Leu ! Ile mutation is associated mainly 

with resistance to TIBO [85,86]. The 106 Val ! Ala mutation mainly leads 

to resistance to nevirapine and HEPT [83,84,87]. The 138 Glu ! Lys 

mutation is responsible for resistance to TSAO [88,89]. The 190 Gly ! Glu 

mutation accounts for resistance to quinoxaline [68], while also leading to a 

dramatic reduction in RT activity [90]; and the 236 Pro ! Leu mutation is 

responsible for resistance to BHAP [91]. 

The rapid emergence of drug-resistant HIV-1 mutants under selective 

pressure of the HIV-1-specific RT inhibitors has been generally viewed as a 

limitation for, if not an argument against, the clinical usefulness of these 

compounds. Yet, several aspects of virus–drug resistance, particularly 

with respect to the NNRTIs, remain to be addressed before the problem 

of resistance can be fully assessed. For example, how pathogenic are drugresistant 

variants in comparison to wild-type virus? How readily are such 

drug-resistant variants transmitted from one person to another? Do virus- 


esistant variants persist when the drug is withdrawn, or do they readily 

revert to the wild type? 

Assuming that the development of drug resistance may indeed 

compromise the clinical usefulness of the NNRTIs, how might this 

problem be prevented or circumvented? If resistance develops to one of 

the NNRTIs, treatment could be switched to any of the other NNRTIs to 

which the virus has retained sensitivity. For example, 5-chloro-3-(phenylsulfonyl)indole-2-carboxamide 

[69] is active against the HIV-1 strains 

that, because of the 103 Lys ! Asn mutation or 181 Tyr ! Cys mutation, 

have acquired resistance to various other NNRTIs (i.e., TIBO, nevirapine, 

pyridinone, BHAP). The a-APA derivative R89439 [67] is active against 

the 100 Leu ! Ile mutant, which is resistant to the TIBO derivatives 

R82913 and R86183. Within the TIBO class, a minor chemical modification, 

the shifting of the chlorine atom from the 9-position (R82913) to the 

8-position (R86183), suffices to restore activity against the 181 Tyr ! Cys 

mutant [92]. Similarly, pyridinone L-702,019, which differs from its 

predecessor L-696,229 only by the addition of two chlorine atoms (in 

the benzene ring) and substitution of sulfur for oxygen (in the pyridine 

ring), remains remarkably active against HIV-1 mutants containing the 

103 Lys ! Asn or 181 Tyr ! Cys mutation [93]. In some instances 

resistance to one of the NNRTIs may even be accompanied by hypersensitivity 

to others: the 236 Pro ! Leu mutation, which causes resistance 

to BHAP, confers 10-fold increased sensitivity to TIBO, nevirapine, and 

pyridinone [91]. 

The 181 Tyr ! Cys mutation, which is responsible for resistance to 

most NNRTIs, has been found to suppress the 215 mutation (Thr ! Phe/ 

Tyr), which is responsible for resistance to AZT [94], and, vice versa, the 

181 Tyr ! Cys mutation can be suppressed by AZT, which thus means that 

the mutations at positions 181 and 215 counteract each other. Yet other 

mutations have proved to counteract each other: 236 Pro ! Leu vs 138 Glu 

! Lys, and, as mentioned, 215 Thr ! Phe/Tyr vs 184 Met ! Val, and 215 

Thr ! Phe/Tyr vs 74 Leu ! Val [47]. Based on the resistance mutations 

that counteract each other, combinations of different drugs could be 

envisaged—namely, combinations of AZT with either TIBO, a-APA, 

HEPT, nevirapine, or pyridinone—and these two drug combinations 

could be extended to three- or four-drug combinations by the addition of 

another ddN analogue (such as 3TC) and/or another NNRTI (such as 

BHAP or TSAO). 

What would seem to be an attractive approach to the prevention of 

resistance development is the ‘‘knocking-out’’ strategy [95]. If NNRTIs, 


such as BHAP (U-88204 or U-90152), are used from the start at a 

sufficiently high concentration (i.e., 1 or 3 AM, respectively), they completely 

suppress virus replication [96,97], with the result that the virus is 

‘‘knocked out’’ and does not have the opportunity to become resistant. If 

U-90152 is combined with AZT, the concentrations of the individual drugs 

can be lowered to achieve total virus clearance [97]. Five NNRTIs (TIBO, 

HEPT, nevirapine, pyridinone, and BHAP) have been shown to ‘‘knock 

out’’ HIV-1 in cell culture when used at concentrations (1–10 Ag/mL) that 

are nontoxic to the cells [95]. That the virus was really knocked out, and 

thus the cell culture cleared (‘‘sterilized’’) from the HIV-1 infection by the 

NNRTIs, was ascertained by two successive rounds of 35-cycle PCR 

(polymerase chain reaction) analysis, which failed to reveal the presence 

of any proviral DNA [95]. Thus, when used at ‘‘knocking-out’’ concentrations, 

the NNRTIs may be expected to effect a long-lasting suppression 

of HIV-1 replication. This ‘‘knocking-out’’ phenomenon could be obtained 

at lower concentrations if the NNRTIs were combined with each 

other, or with any of the ddN analogues (i.e., AZT), particularly if selected 

on the basis of the ‘‘mutually counteracting mutation’’ principle. 

VII. CONCLUSION 

An acute HIV infection can be blocked at any of the following stages of 

the infection: virus adsorption, virus–cell fusion, viral uncoating, and reverse 

transcription. At the reverse transcriptase (RT) level, chemotherapeutic 

intervention could be envisaged at either the substrate or a non– 

substrate binding site. Polyanionic substances (i.e., sulfated polysaccharides) 

prevent virus adsorption; plant lectins, succinylated (or aconitylated) 

albumins, and triterpene (i.e., betulinic acid) derivatives interfere 

with virus–cell fusion; bicyclams inhibit viral uncoating; 2V,3V-dideoxynucleosides 

(ddNs) and acyclic nucleoside phosphonate analogues, following 

intracellular conversion to their phosphorylated derivatives, interact with 

the substrate binding site of the RT; and the nonnucleoside reverse transcriptase 

inhibitors (NNRTIs) are targeted at a non–substrate binding site 

of HIV-1 RT. Some of these compounds (viz., bicyclams) and, among the 

NNRTIs, some of the HEPT and a-APA derivatives, were found to inhibit 

HIV-1 replication at concentrations (f1 ng/mL) that were 100,000-fold 

or more below the cytotoxicity threshold. As a rule, it may be postulated 

that the more specific the antiviral action, the more likely the development 

of virus–drug resistance; hence, NNRTIs, which engage in a highly 


specific interaction with HIV-1 RT, rapidly lead to the emergence of drugresistant 

virus strains. To prevent such drug-resistant virus strains from 

emerging, several strategies could be envisaged, the most attractive being 

the combination of several drugs at concentrations high enough to ‘‘knock 

out’’ the virus from the start. This ‘‘knocking-out’’ phenomenon has been 

achieved with the NNRTIs, regardless of whether combined with any of 

the ddN analogues, and it may be extended to combinations of drugs that 

interact at targets other than the reverse transcriptase. 


The original investigations of the author are supported by the Biomedical 

Research Programme of the European Community, the Belgian 

Nationaal Fonds voor Wetenschappelijk Onderzoek, the Belgian Fonds 

voor Geneeskundig Wetenschappelijk Onderzoek, the Belgian Geconcerteerde 

Onderzoeksacties, and the Janssen Research Foundation. I 

thank Christiane Callebaut for her dedicated editorial assistance. 

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