Trekking Through Receptor Chemistry - Università degli Studi di ...

28 th CAMERINO-CYPRUS-NOORDWIJKERHOUT SYMPOSIUM
Trekking Through Receptor Chemistry
Camerino, May 16-20, 2010
UNIVERSITÁ DEGLI STUDI DI CAMERINO
Palazzo Ducale, Sala della Muta

Cover image
Engraving of a view of Camerino taken from the book ”Theatrum Celebriorum Urbium”,
Amsterdam 1652

28 th CAMERINO-CYPRUS-NOORDWIJKERHOUT
SYMPOSIUM
Trekking Through Receptor Chemistry
Organized by the
University of Camerino
under the sponsorship of the
Medicinal Chemistry Division of the Italian Chemical Society (SCI)
and the
European Federation for Medicinal Chemistry (EFMC)
Camerino, Italy
May 16-20, 2010
UNIVERSITÁ DEGLI STUDI DI CAMERINO
Palazzo Ducale, Sala della Muta

Organizing Committee
Ambrosini D.
Angeli P.
Del Bello F.
Giannella M.
Giardinà D.
Giorgioni G.
Piergentili A.
Pigini M.
Quaglia W.
Index
History 3
Programme 5
Opening 15
Introductions to the Sessions 19
Lectures 29
Short Communications 97
Poster Session 111
List of Participants 138
Acknowledgments 143
Scientific Committee
Donati D. (Italy)
Gaviraghi G. (Italy)
Giannella M. (Italy)
Glennon R.A. (USA)
Gualtieri F. (Italy)
Leurs R. (The Netherlands)
Makriyannis A. (Cyprus)
Melchiorre C. (Italy)
Mosti L. (Italy)
Pellicciari R. (Italy)
Ratti E. (Italy)
Timmerman H. (The Netherlands)
Triggle D.J. (USA)

HISTORY
The interplay between molecular structure and biological activity is central to contemporary biomedical
and translational research, relevant not only to structural biologists, but also to diverse scientists
involved in the study of bioactive compounds and signaling mediators and those working in drug
discovery and development. International meetings on this topic have been held over the past thirty
years. In 1977, Dutch medicinal chemists organized a symposium, “Trends in Medicinal Chemistry,”
which became known as the Noordwijkerhout Symposium. The symposium emphasized the
increasingly interdisciplinary nature of the then-emerging field of structural biology. The second and
third Noordwijkerhout Symposia took place in 1981 and 1985, respectively. Contemporaneously, in
1978, a team of medicinal chemists from the University of Camerino, with the sponsorship of the Italian
Chemical Society, organized a Camerino meeting on the synergy between the chemical and biological
aspects of receptor research. This symposium became the first of a series known as the Camerino
Symposia on Receptor Chemistry, the second of which convened in 1983.
In 1987, the growing number of symposia risked redundancy. To eliminate this risk and enhance further
the symposium content, the organizers of the Noordwijkerhout and Camerino symposia decided to
cooperate closely. As a result of this concerted international effort between the Netherlands and Italy,
every two years since 1987 a joint Camerino-Noordwijkerhout Symposium has been held under the
auspices of the European Federation of Medicinal Chemistry in either Camerino (1987, 1991, 1995,
1999, 2003, and 2007) or Noordwijkerhout (1989, 1993, 1997, 2001, and 2005).
Likewise under the auspices of the European Federation of Medicinal Chemistry, the Cyprus
Conference was first convened in Limassol, Cyprus, in 1983. All ten Cyprus conferences have been
organized by a group of internationally renowned medicinal chemists for the purpose of emphasizing
state-of-the-art research and approaches in drug discovery. These meetings covered a wide range of
disciplines, including combinatorial, biophysical, and computational chemistry; molecular and structural
biology; the “-omics” technologies; informatics; and new targets for medicinal chemistry. The program
was designed for scientists and research administrators who seek to exploit novel opportunities in drug
discovery. The conference format and seaside locale encourage interaction among all participants. The
Cyprus Conference has now joined the Camerino-Noordwijkerhout group to constitute the first tripartite
European meeting in medicinal chemistry. In 2008 the 26th Cyprus-Noordwijkerhout-Camerino
Symposium in this long lasting series was held in Limassol (Cyprus) and in 2009 the 27th edition in
Noordwijkerhout (The Netherlands).
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4
Agreement on a cooperation between the organizers of the series of symposia known as the
The responsible organizers confirm that
Camerino-Cyprus-Noordwijkerhout symposia.
- they will each organize a medicinal chemistry symposium in subsequent years, to start in 2007
with a symposium in Camerino, 2008 a Cyprus-symposium, 2009 in Noordwijkerhout, 2010 in
Camerino and so on (spring season);
- each symposium will have its own specific character: Camerino focusing on Advances and
Perspectives in Receptor Research, Cyprus on New Approaches and Methods in Drug Research,
Noordwijkerhout on Trends in Medicinal Chemistry;
- the Organizing Committees of the individual symposia will be responsible for the organization
of ‘their’ symposia in all aspects, including programme and finances;
- the Organizing Committee for each conference will include at least one member from each of
the other two;
- the local Organizing Committees will exchange their mailing lists for the purpose of promoting
the symposia;
- the symposia will be announced sharing the same logo and the common name “Cyprus-
Noordwijkerhout-Camerino”. The first name indicates the location where the symposium takes
place and the sequence of the series is indicated by the order of the names;
- the three organizing committees guarantee to each other they will organize a symposium in the
indicated year as follows from the sequence of the series;
- the conference will continue seeking sponsorship from EFMC.
Signed by
Representative Camerino (Prof. Mario Giannella)
Representative Cyprus (Prof. Alexandros Makriyannis)
Representative Noordwijkerhout (Prof. Henk Timmerman)

PROGRAMME
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6
Panorama of Camerino
The shrine of Macereto

SUNDAY, MAY 16
Sala della Muta, Palazzo Ducale
18:45-19:00
M. GIANNELLA
Opening
19:00-19:30
U. HACKSELL
Discovery and development of drugs for CNS diseases
19:30-20:15
Welcome of ANTONIO DAL SORDO TRIO
OSTERIA DI MEZZO
20:30 Welcome Dinner
CORTE DEL PALAZZO DUCALE
Ancient and Modern Emotions
Book exhibition by Treccani
Lighting effects by Guzzini
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8
MONDAY, MAY 17
Sala della Muta, Palazzo Ducale
Starting on the right foot: selection criteria in lead discovery
9:00-9:10
Chairman: R. J. Thomas
9:10-9:40
R. E. HUBBARD
Too much choice: selecting which fragments to optimise
9:40-10:10
E. H. KERNS
Integration of drug-like properties in selection, optimization and biological
assessment in lead discovery
10:10-10:30 Coffee Break
10:30-11:00
C. GHIRON
The design, management and maintenance of a screening collection
11:00-11:30
P. D. LEESON
Can we improve “Compound Quality” ?
11:30-11:45
I. IJJAALI
SAR knowledgebase approach for exploring target-ligand selectivity
patterns: application to kinase target family
13:00 Lunch (Hotel I Duchi)

MONDAY, MAY 17
Sala della Muta, Palazzo Ducale
New avenues to target G protein coupled receptors
15:00-15:10
Chairman: R. Leurs
15:10-15:40
M. BOUVIER
Ligand-based GPCR signalling; potential implications for drug discovery
15.40-16:10
G. J. R. ZAMAN
New avenues to target Wnt/Frizzled receptor signaling
16:10-16:40
M. ALLEGRETTI
Pharmacological characterization of functionally selective CXCR inhibitors
16:40-17:00 Coffee Break
17:00-17:30
M. J. SMIT
Nanobodies as new GPCR modulators
17:30-18:00
S. SCHANN
mGluR4 Positive allosteric modulator: new paradigm for CNS indication
treatment
18:00-18:30
R. PELLICCIARI
Prospects for a TGR5-mediated control of glucose homeostasis: focus on the
clinical candidate S-EMCA (INT-777)
18:30-18:45
S. COSTANZI
Identification of GPCR ligands through docking-based virtual screening: not
only crystal structures but also homology models
19:30 Dinner (Hotel I Duchi)
21:00 Offerta dei Ceri (Basilica S. Venanzio)
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TUESDAY, MAY 18
Sala della Muta, Palazzo Ducale
G protein coupled receptors: old landscape with new eyes
9:00-9:10
Chairman: D. Donati
9:10-9:40
C. ENZENSPERGER
The dopamine D1-family: steps towards selectivity
9:40-10:10
A. MAKRIYANNIS
The endocannabinoid proteins as drug targets
10:10-10:30 Coffee Break
10:30-11:00
D. PIOMELLI
The endocannabinoid system and the regulation of pain and emotion
11:00-11:30
S. RONZONI
Novel potent and selective ORL-1 antagonists with efficacy in animal models
of Parkinson’s disease and neuropathic pain
11:30-12:00
P. S. PORTOGHESE
Are heteromeric G protein-coupled receptors relevant to in vivo
pharmacology? Opioid receptors as a case in point
12:00-12:15
R. G. BOOTH
Functionally-Selective Serotonin 5-HT2 GPCR Drugs:
5-HT2C agonists with 5-HT2A/2B inverse agonist activity for neuropsychiatric
disorders
12:15-12:30
R. A. SMITS
Medicinal chemistry of novel histamine H4 receptor antagonists
13:00 Lunch (Hotel I Duchi)

TUESDAY, MAY 18
Sala della Muta, Palazzo Ducale
14:45-15:00
R. FORLANI
The dark side of research: multiple data source management
Application of computational methods in lead discovery
15:00-15:10
Chairman: A. Martinelli
15:10-15:40
M. S. CONGREVE
Stabilized GPCRs for structure based drug design
15:40-16:10
K.-H. BARINGHAUS
Computer-assisted lead finding of GPCR modulators
16:10-16:25
A. CARRIERI
Novel α1-adrenoreceptor antagonists related to openphendioxan: biological
evaluation and α1d computational study
16:25-16:40
T. TUCCINARDI
Protein kinases: docking and homology modeling reliability
16:40-17:00 Coffee Break
Receptors and beyond
17:00-17:10
Chairman: R. A. Glennon
17:10-17:40
P. J. L. SVENNINGSSON
Regulation of 5-HT receptor functions by p11
17:40 18:10
M. K. SCHULTE
Allosteric modulation of neuronal nicotinic acetylcholine receptors
18:10-18:40
K. MOHR
Dualsteric GPCR-targeting: fine-tuning binding selectivity and signalling
pathway activation
18:40-18:55
M. DUKAT
A negative allosteric modulator of α7 nAChRs
19:30 Typicality & Folklore (Castello di Lanciano)
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WEDNESDAY, MAY 19
Sala della Muta, Palazzo Ducale
Polypharmacology: creating selective non selectivity
9:00-9:10
Chairmen: M. L. Bolognesi and C. Melchiorre
9:10-9:40
J. S. MASON
Polypharmacology: using it to differentiate and as an advantage with
selective non-selectivity
9:40-10:10
R. MORPHY
Multi-target drug discovery – past, present and future
10:10-10:30 Coffee Break
10:30-11:00
C. J. VAN DER SCHYF
Multiple mechanism drugs for neurodegenerative diseases and post-stroke
neuroprotection
11:00-11:30
M. B. H. YOUDIM
Novel therapeutic approaches constituting multifunctional neuroprotective
and neurorestorative drugs for neurodegenerative diseases
11:30-11:45
X.-J. LI
Chemoinformatics approaches for anti-dementia traditional Chinese
medicine research and implications for finding polypharmacology drugs
11:45-12:00
E. SIMONI
Curcumin analogs as multi-target antioxidants: focus on mithocondria
13:00 Lunch (Hotel I Duchi)

WEDNESDAY, MAY 19
Sala della Muta, Palazzo Ducale
Orexin antagonists as novel treatment of insomnia
15:00-15:10
Chairman: H. Timmerman
15:10-15:40
E. MERLO PICH
Central orexin peptidergic system as target for novel treatments of insomnia,
anxiety and drug addiction: recent findings from the GSK Discovery
Performance Unit
15:40-16:10
R. DI FABIO
Bis-amido piperidine derivatives as in vitro and in vivo potent dual orexin
receptor antagonists
16:10-16:40
A. J. ROECHER
Discovery of potent, diazepane-containing dual orexin receptor antagonists
for the treatment of insomnia
16:40-17:00 Coffee break
17:00-17:30
C. BOSS
Orexin receptor antagonists – A new therapeutic principle in CNS
disorders?
17:30-17:45
J. BENTLEY
Identification of a novel OX1 antagonist series by high-throughput screening
and SDM-directed homology modeling of orexin receptors
17:45-18:00
J. SELENT
Sodium ions toggle the rotamer switch in sodium-sensitive dopaminergic Gprotein
coupled receptors
18:00-19:00
POSTER SESSION
19:30 Dinner (Hotel I Duchi)
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THURSDAY, MAY 20
Sala della Muta, Palazzo Ducale
Application of stem cell technology to drug discovery
9:00-9:10
Chairman: A. Makriyannis
9:10-9:40
F. FAROUZ
Novel oxysterols activate the hedgehog pathway in vitro and stimulate spinal
fusion in vivo
9:40-10:10
P. TUNICI
Brain tumor stem cells as a tool to identify novel therapeutics
10:10-10:30 Coffee Break
10:30-11:00
R. J. THOMAS
Successful modulation of tumor stem cells with small molecule hedgehog
pathway antagonists
11:00-11:30
G. GAVIRAGHI
Therapeutic modulation of the Wnt signalling pathway, an innovative
approach for oncology and neurodegeneration
11:30-12:00
Looking forward
M. GIANNELLA, A. MAKRIYANNIS, H. TIMMERMAN
12:00-12:30
D. J. TRIGGLE
The sustainability of pharmaceuticals in the 21 st century: the idle thoughts of
an idle fellow
13:00 Lunch (Hotel I Duchi)
15:00 Social tour
Matelica - Visit to “Museo Archeologico” and “Museo Piersanti”
20:00 Social Dinner (Villa Fornari)

OPENING
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Notes

DISCOVERY AND DEVELOPMENT OF DRUGS FOR CNS DISEASES
Uli Hacksell
ACADIA Pharmaceuticals Inc., San Diego, California, USA
Current antipsychotic drugs suffer from various shortcomings: they are relatively effective in
treating the positive symptoms of schizophrenia, but remain largely ineffective in treating the
negative symptoms and, in particular, the cognitive deficits. In addition, their side effect
burden is a major problem. ACADIA is applying modern drug discovery technologies
integrated with traditional methods in attempts to discover novel antipsychotic agents. Here I
will discuss our efforts to achieve both antipsychotic and procognitive activity by selective
stimulation of M1 muscarinic receptors.
We have access to a proprietary functional assay technology that allows us to study a broad
range of GPCR interactions. Having enabled a functional ultra-HTS assay for M1 agonism, we
screened our small-molecule library for M1 agonists and discovered a novel type of muscarinic
agonist, AC-42, that selectively stimulates M1 receptors. AC-42 does not bind to the orthosteric
site of the M1 receptor, but binds to an allosteric region of the receptor that is non-conserved
between the five muscarinic receptor subtypes. We have now developed several series of more
potent and drug-like M1-selective agonists, including AC-260584.
As part of our effort to develop novel concepts in schizophrenia therapy, we profiled the GPCR
interactions of known antipsychotic agents and their relevant metabolites and found that
NDMC, the key-active metabolite of clozapine, is a partial M1 receptor agonist and that NDMC
may be responsible for many of the uniquely positive effects of clozapine. We believe that the
combination of D2 antagonism, 5-HT2A inverse agonism and selective M1 muscarinic agonism
in the same molecule has the potential to provide superior antipsychotic activity. Several
compounds with this profile have been discovered. These pro-cognitive antipsychotic drug
candidates (PCAPs) are active in preclinical antipsychotic and cognition models.
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18
Trekking through the “Cresta della Sibilla”
Palazzo Borghese

INTRODUCTION TO THE SESSIONS
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STARTING ON THE RIGHT FOOT: SELECTION CRITERIA IN LEAD
DISCOVERY
Russell J. Thomas
Medicinal Chemistry Department, Siena Biotech S.p.A., Strada del Petriccio e Belriguardo, 35, 53100, Siena, Italy
rthomas@sienabiotech.it.
The quality of structural starting points in any integrated drug discovery programme will have an enormous impact
on the activities, costs and ultimately chances of success of the project.
The more time and money spent on radical modifications of chemical starting points, attempting to transform
target affinity into efficacy and selectivity, the lower the probability of finally arriving in the clinic with an
effective, innovative and safe medicine (1).
This session looks at a number of complementary approaches to “starting on the right foot” with high quality
compounds and data-driven decision tools as early as possible in a project.
Optimizing compound affinity often results in an increase in molecular weight and lipophilicity. To avoid this
tendency can be extremely challenging. Fragment based approaches accept this fact and work around it by
carefully selecting significantly smaller ligands (2), albeit with often modest potency, and then attempt to improve
the potency and properties through structure-based design.
Irrespective of whether the initial lead structures are derived from fragments or larger compounds, research
projects require clear vision of the importance of in vivo efficacy and safety as opposed to in vitro affinity (3).
Additionally, a suite of high quality in silico, in vitro and in vivo models (4), with appropriate tools to collate the
data generated, are essential to build new hypotheses to test.
Success depends not only upon careful selection of what to screen and synthesize but also on the way these
compounds are characterized, organized, archived and distributed to projects. Many aspects of how this is
achieved will depend on the size and nature of the research organization however the basic scientific and
organizational principles remain.
(1) Proudfoot, J. R., Bioorg. Med. Chem. Lett., 2002, 12, 1647-1650.
(2) Chen, I-J., Hubbard, R. E. J. Comput. Aided Mol. Des., 2009, 23, 603-620
(3) Leeson, P. D., Springthorpe, B. Nature Rev. Drug Discov., 2007, 6, 881-890
(4) Kerns, E., Di, L. Drug-like Properties: Concepts, Structure Design and Methods: from ADME to Toxicity Optimization,
Academic Press, 2008.
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NEW AVENUES TO TARGET G-PROTEIN COUPLED RECEPTORS
Rob Leurs
Department of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, VU University Amsterdam, the
Netherlands.
G-protein coupled receptors have for many years been one of the most favorite drug targets for effective
therapeutic intervention in many disorders. Small molecules have for many years been very effective tools to
target this important class of receptor proteins. The recent discovery of many new concepts of G protein coupled
receptor action (allosterism, ligand-biased signaling, heterodimerization) have greatly increased the possibilities to
affect G protein coupled receptor function with small molecules. Moreover, research into GPCRs targeting has
been growing at a fast pace and the range of approached that can be applied to target GPCRs continues to grow. In
this session various new ways to target GPCRs will be discussed.
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G PROTEIN COUPLED RECEPTORS: OLD LANDSCAPE WITH NEW
EYES
Daniele Donati
Nerviano Medical Sciences srl, Oncology Business Unit, Viale Pasteur 10, 20014 Nerviano (MI), Italy
The G-protein coupled receptors are seven trans-membrane domain (7TM) proteins and represent the largest
family of proteins in the human genome. They are involved in the modulation of a large number of physiological
and pathophysiological processes, and are the target for about 30% of prescription drugs (1-3). More in general,
GPCRs represent the most explored and the successful target family in drug discovery, covering a number of
therapeutic areas, and many new drug candidates are being reported as selective ligands for such receptors (4).
With very few exceptions, all the currently available therapeutics based on the relevant receptors are targeting their
orthosteric binding sites and this reflects the nature of the assays (typically binding assays) generally utilized as
primary screening for novel chemical entities(5).
In recent years, the need for more selective and safer therapeutic agents has triggered new research efforts aimed at
the intimate understanding of the mechanism of action of GPCRs, with specific attention to the signal transduction
mechanisms as well as to the nature of the effective receptor system responsible for the observed physiological
action of both endogenous and exogenous ligands. A great deal of interest has indeed emerged for allosteric
modulation (positive and negative) of both homodimeric and heterodimeric GPCR receptors (5, 6). The number of
experimental evidences supporting the existence and the relevance of GPCR oligomerization on drug discovery is
growing and it is now evident the need for new strategies in the search of novel and more effective drugs targeting
GPCR receptors. We need indeed to consider that different and novel screening approaches have to be used (eg by
co-expressing the protomers in the same cell line). As well, we need to consider that the oligomerization may be
tissue specific and therefore we may need to explore more in deep the tissue selectivity for a new GPCR ligand.
(1) Lagestrom, M.C.; Schioth, H.B. Nat. Rev. Drug Discov. 2008, 7, 339-357.
(2) Overington, J.P. Nat. Rev. Drug Discov. 2006, 5, 993-996.
(3) Jacoby, E.; Bouhelal, R.; Gerspacher, M.; Seuwen, K. Chem MedChem 2006, 1, 761-782
(4) Gilchrist, A. Curr. Opin. Drug Discov. 2008, 3, 375-389
(5) Milligan, G., Smith, N.J. Trends Pharmacol. Sci. 2007, 28, 615-620 and references cited therein
(6) Panetta, R.; Greenwood, M.T., Drug Discov. Today 2008, 13, 1059-1066 and references cited therein
I-03

APPLICATIONS OF COMPUTATIONAL METHODS IN LEAD
DISCOVERY
Adriano Martinelli
Department of Pharmaceutical Sciences - University of Pisa.
At present it is largely accepted that computational methods are an essential tool for discovering new
pharmacological leads.
The lectures of this section concern computational studies of biological targets, which is the first step for a
structural based discovery strategy able to clarify the ligand-receptor interactions and to furnish the bases for the
virtual screening of new ligands.
Three lectures deal with G-protein-coupled receptors (GPCRs). The superfamily of GPCRs are single polypeptide
chains possessing seven hydrophobic transmembrane-spanning segments that couple with an effector molecule
through a trimeric G protein complex. At present they are the target of about 50% of the drugs in the market.
Baringhaus et al. propose two approaches for finding GPCR modulators, the first one is based on the quantitative
similarity description of GPCR pairs, the latter on the definition of structure based pharmacophore models.
A knowledge of the GPCR 3D structure could be of great help in the task of understanding their function and in
the rational design of specific ligands, however high-resolution structural characterization is still an extremely
difficult task. For this reason, great importance has been placed on molecular modeling studies, and especially on
homology modelling (HM) techniques which, however, request experimentally determined structures of suitable
templates.
Congreve describes a new technology able to stabilize GPCRs outside of the cell membrane therefore allowing
their crystallization and X-ray structure determination.
Carrieri et al. show how a computational study can help to elucidate the interaction with the receptor of novel α1-
adrenoreceptor antagonists.
The topic of the last lecture from Tuccinardi concerns an analysis of the reliability of molecular docking and
homology modeling methods applied to another important class of drug targets, the protein kinases.
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RECEPTORS AND BEYOND
Richard A. Glennon
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia 23298 USA
Paul Ehrlich and John Langley, contemporaneously investigating the effects of chemical agents or plant extracts
on physiological function in the late 1800s, suggested that specific interactions might be occurring. Ehrlich
proposed the “side chain theory” (i.e., cells might have an affinity for certain chemicals or toxins because of their
chemical composition), and in 1905 Langley introduced the term “receptive substance”. In the 1920s and 1930s
Clarke and Gaddum introduced the concepts of log dose-response curves, competitive antagonism, and developed
equations that might explain drug receptor interactions. Schild mathematically described, and demonstrated the
concepts of dose-ratio and affinity. Subsequent contributions include those made by Ariens (partial agonists,
intrinsic activity), Stephenson (efficacy), Furchgott (spare receptors, intrinsic efficacy), Paton (rate theory), del
Castillo and Katz (interconvertible forms of receptors), Lefkowitz et al., (ternary complex model), Black and Leff
(operational model), Costa and Herz (constitutive activity), Lefkowitz and Costa (allosteric ternary complex
models), Weiss/Kenakin (cubic ternary complex model), and others. From its humble beginnings, the “receptive
substance” has become more complex. At least four major superfamilies of receptors are recognized: ion channel,
G-protein coupled (GPCRs), tyrosine kinase (Trk), and nuclear receptors. Furthermore, the >1,000 GPCRs are
further divided into six classes: Class A (rhodopsin-like), Class B (secretin family), Class C (metabotropic), Class
D (fungal), Class E (cyclic AMP), and Class F (frizzled). And, many of these receptors have been targets of drug
discovery/development.
Apart from direct interactions with a receptor, there are other receptor-associated targets that are of interest
because they can indirectly influence the actions of a given neurotransmitter. Several such possibilities include:
allosterism, modulation of post-receptor events, and agonist-directed trafficking. i) An allosteric (or allotopic)
effect results when an agent modifies the effect of another (i.e., orthosteric agent) by binding to a protein at a site
(i.e., allosteric site) topologically different from that of the orthosteric ligand. Allosteric modulators may be
positive or negative. Early studies focussed on allosteric regulation of ion channel receptors, but more recently are
being directed to GPCRs. ii) Dopamine (DA) is associated with a cAMP/PKA second messenger pathway that
targets DA- and cAMP-regulated phosphoprotein (i.e., DARPP-32). The function of DARPP-32 is regulated by its
phosphorylation state, and agents that target receptors other than DA receptors can influence the phosphorylation
state of DARPP-32 to indirectly influence its effects. iii) Certain receptors can be coupled to multiple second
messenger systems. Hence, the possibility exists that different agonists might stabilize different agonist receptor
conformations to result in selective activation of one second messenger system over another. These topics are the
focus of the present session.
I-05

POLYPHARMACOLOGY: CREATING SELECTIVE NON-
SELECTIVITY
Carlo Melchiorre and Maria Laura Bolognesi
Department of Pharmaceutical Sciences, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna,
Italy.
Drug discovery has contributed greatly to advancing the life sciences and the wellbeing of society in the past
century. However, several diseases remain incurable and we are facing an unpredictable productivity crisis. In the
last 20 years, while the costs of R&D have steadily increased, there has been a gradual decline in its productivity,
as measured by the number of new approved molecular entities. This has been attributed to drug discovery’s
gradual move from an entirely human phenotype-based endeavor to the so-called ‘reductionist approach’. This
approach attempts to reduce drug action to the level of individual genes, single proteins, and one potential
modulating molecule. Thus, the ‘one gene, one target, one drug’ paradigm seeks to generate potent and exquisitely
selective ligands that could guard against unwanted side effects. 1
However, it is now widely accepted that the majority of diseases that threaten humanity are multifactorial, with
substantial environmental and genetic components. For this reason, a selective single-target drug might lack
efficacy, while a treatment able to address this complexity will have a greater chance of success. 2 To this end,
there has been growing recognition that polypharmacology might provide therapeutic benefits where traditional
single-target drugs have failed. A combination of drugs, which offers the prospect of additional benefits, is being
used to treat several complex diseases such as HIV and hypertension. A more recent interpretation of
polypharmacology considers single chemical entities able to simultaneously modulate several molecular targets. 3
Although these concepts emerged less than 10 years ago, multitarget kinase inhibitors are already a reality in
cancer therapy. 4 Their introduction into the market provided the pharmaceutical community with the proof of
concept that a ligand with a rationally designed multimodal mechanism of action would not have intrinsically
overwhelming toxicity. Other successful examples of multitarget drugs combining high efficacy with reasonable
safety are emerging in the fields of neurodegeneration 5 and depression. It is highly conceivable that several others
will be identified in the near future, bridging the conceptual gap between specific and nonselective drugs.
(1) Hopkins, A. L.; Mason, J. S.; Overington, Curr. Opin. Struct. Biol. 2006, 16, 127-136.
(2) Kong, D. X.; Li, X. J.; Zhang, H. Y. Drug Discov Today 2009, 14, 115-119.
(3) Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini, M.; Melchiorre, C. J. Med.
Chem. 2008, 51, 347-372.
(4) Morphy, R. J. Med. Chem. 2009, 53, 1413-1437.
(5) Van der Schyf, C. J.; Youdim, M. B. Neurotherapeutics 2009, 6, 1-3.
I-06
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26
OREXIN ANTAGONISTS AS NOVEL TREATMENT OF INSOMNIA
Henk Timmerman
VU University Amsterdam
(henktim @ planet.nl)
Ever since the first symposium of this series took place in Camerino the focus has been on receptors, their
mechanisms, ligands, structure and role in pharmacotherapy. As a consequence certain classes of medicines
could in the “early” hardly get attention as their molecular targets were just unknown.; antiepileptics, anti obesity
agents, sleep pills, to mention a few. In the 1987 symposium out of the twenty lectures four dealt with adrenergic
receptors, five with cholinergic and three with histamine receptors.
Much has changed since than. The introduction of molecular biology in medicinal chemistry and pharmacology
made it possible to identify the targets for several well known medicines for which a target was unknown.
Moreover, a number of new targets were found , new medicines could be envisaged.
An example of a new class of targetable receptors is the given by the receptors belonging to the orexin system.
The orexins were first described in the late 1990ies and subsequently found to bind to a n organ GPCR. The
receptors have been seen as implicated in the regulation of the intake of food. Currently however the focus is
mainly on sleep disorders,.
During this symposium Merlo Pich will introduce the biology orexin system; subsequently ,three lectures will
deal with new compounds, under investigation for use in the treatment of insomnia.
I-07

APPLICATION OF STEM CELL TECHNOLOGY TO DRUG
DISCOVERY
Alexandros Makriyannis, Ph.D.
George D. Behrakis Chair in Pharmaceutical Biotechnology
Director, Center for Drug Discovery, Northeastern University
Stem cell technologies have taken an important place in therapeutics. The most advanced efforts are in the fields
of oncology and neurodegeneration. The three contributions to this symposium will discuss current efforts in that
direction. Tumor stem cells can be used as tools for developing new medications. Additionally, small drug-like
molecules are designed to modulate stem cell-related signaling.
I-08
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28
Fiastra Lake
Pilato’s Lake

LECTURES
29

30
Notes

TOO MUCH CHOICE: SELECTING WHICH FRAGMENTS TO
OPTIMISE
Roderick E. Hubbard
University of York and Vernalis Ltd.
Over the past eight years, we have developed and applied fragment-based ligand discovery methods to identify
compounds that bind to a range of protein targets. These approaches have been developed both in a drug
discovery (Vernalis) and an academic (York) context. Some of these compounds have been developed into
effective inhibitors to probe the mechanism of action of certain protein targets. Others have been optimized to
provide clinical candidates, now in clinical trials. Fragments are just small, weak hits. The main challenges are
design of the library, robust identification of which fragments bind and the need for structural information to
decide how and which fragments to progress. The advantages are that a small library can sample a potentially
large chemical diversity to generate novel lead compounds and that hits can be identified for new classes of target
for which existing compound collections cannot provide a hit. Various approaches have been used to develop the
fragment hits into useful leads. These include (a). linking fragments together, (b). growing by directed limited
library synthesis or searching for nearest neighbours in the accessible compound databases, or (c). merging where
the structures of fragments, existing tool compounds and virtual screening hits provides guidance for how to
merge features from different compounds together. These experiences will be summarised.
One of the big challenges is deciding which fragments to take forward. The methods have quite a high hit rate -
for well-defined binding sites, up to 100 hits can be generated from a library of 1200 fragments. In this
presentation I will discuss what properties could be used to decide between fragments, including issues of
chemical tractability, vectors, opportunities to achieve selectivity and how best to integrate this information with
biophysical insights from binding assays and measurements of the kinetics (SPR) and thermodynamics (ITC) of
binding. There is no simple answer - like most areas of drug discovery, it can be difficult to make the right
decisions.
References:
(1) Hubbard, R.E. et al , (2007) "The SeeDs approach: Integrating Fragments into Drug Discovery", Current Topics
Medicinal Chemistry, 7, 1568-1581
(2) Brough, P.A. et al (2009), “Combining hit identification strategies: Fragment-based and in silico approaches to orally
active 2-aminothieno[2,3-d]pyrimidine inhibitors of the Hsp90 molecular chaperone”, J Med Chem, Epub 17th July
(3) Chen, I and Hubbard, R.E. (2009), “Lessons for fragment library design: analysis of output from multiple screening
campaigns”, J Comp Aided Mol Des, Epub Jun 3
(4) Fisher, M and Hubbard, R.E. (2009), “Fragment-based ligand discovery”, Mol Interventions, 9, 22-30
(5) Schulz, M N and Hubbard RE (2009), “Recent progress in fragment-based lead discovery “, Curr Opin
Pharmacology, Epub May 27
L-01
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Notes

INTEGRATION OF DRUG-LIKE PROPERTIES IN SELECTION,
OPTIMIZATION AND BIOLOGICAL ASSESSMENT IN LEAD
DISCOVERY
Edward H. Kerns
Wyeth Research, CN8000, Princeton NJ08543-8000, U.S.A.
ADME/Tox properties are a major focus of drug discovery. They determine the ultimate delivery properties of the
drug product from the dosage form to the therapeutic target and its toxicity. Thus, compound properties affect the
efficacy and safety of the drug. For this reason, drug-like properties are integrated in lead selection and
optimization with other major objectives (e.g., activity, selectivity, novelty). In addition, properties affect
biological experiments: solubility, chemical stability and permeability affect compound delivery to the target in
HTS, enzyme/receptor and cell-based assays. This presentation will discuss key ADME/Tox properties and their
assessment, effects and improvement in Lead Discovery.
L-02
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34
Notes

THE DESIGN, MANAGEMENT AND MAINTENANCE OF A
SCREENING COLLECTION
Compound Management and Analytical Services in the Small Pharma
Chiara Ghiron, Raffaella Brandi, Eva Genesio
Siena Biotech SpA, Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy
The organisation of compound acquisition, management, logistics and analytical services in the small pharma is
deceptively simple. While the number of compounds in the compound collection may not be as large as in bigger
companies, the issues related to compound collection creation, sample preparation and distribution within the site,
and analytical quality controls are essentially the same.
In the presentation we will show how the process is organised in Siena Biotech, illustrating our compound
acquisition and registration procedures, internal requests organisation, distribution of assay-ready plates to
screening units, and the analytical controls in place at the different stages of the drug discovery process.
We will also illustrate how we improved the process over the years, after thorough analysis of past practices and
through implementation of in-house developed software where necessary.
L-03
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36
Notes

CAN WE IMPROVE ‘COMPOUND QUALITY’?
Paul D. Leeson
AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leics, LE11 5RH, UK
Following the ‘rule of 5’ publication in 1997 there have been further studies which have underlined the importance
of fundamental physical properties in controlling permeability, metabolic stability, organ toxicity, binding to the
hERG channel, and receptor promiscuity. Extending the ‘rule of 5’, these recent findings have introduced more
stringent physical property ranges for risk reduction in drug discovery (1-6).
Comparisons of oral drugs launched before and after 1983 reveals that several drug-like properties, for example
lipophilicity and % polar surface area, are not changing over time, whereas others, for example molecular weight
and hydrogen bonding atoms, are increasing in newer drugs; time-constant properties are suggested to be more
important success factors in drug development (7). The physical properties of current medicinal chemistry are
reflected by the patent literature, where increases in all molecular properties vs. current drugs are evident (1),
suggesting significantly increased ADMET risk in pharmaceutical company portfolios. Moreover, there are
meaningful differences between pharmaceutical company physical property profiles, indicating that drug
properties are influenced at least as much, or more, by local lead generation and optimisation strategy, culture and
behaviour than by specific target difficulty. This conclusion is supported by a comparative analysis of company
target-specific patenting practices over time.
Ligand lipophilicity efficiency (LLE = p(Activity) – LogP/D) is proposed as a key measure of compound quality.
Increasing LLE to >5 (ie potency

38
Notes

LIGAND-BASED GPCR SIGNALLING; POTENTIAL IMPLICATIONS
FOR DRUG DISCOVERY
Michel Bouvier
Department of Biochemistry, Groupe de Recherche Universitaire sur les Médicaments, Institute for Research in Immunology
and Cancer, Université de Montréal, Montréal, Québec, Canada
Traditionally known for their ability to selectively activate a unique hetero-trimeric G protein, individual G
protein-coupled receptors (GPCR) have since been shown to activate multiple G protein subtypes as well as G-
protein independent signalling cascades. In addition different ligands were found to selectively promote the
engagement of distinct signalling partner subsets of a given receptor. Some compounds were also found to have
clearly distinct, some time, opposite efficacies toward different pathways engaged by the same receptor. This
phenomenon known as ligand-biased signalling offers interesting opportunities to develop compound with
increased selectivity profiles but present important challenges for the drug discovery process in particular in the
context of high throughput screening. To better understand the molecular mechanisms directing ligand-biased
signalling we developed new assays based on luminescence and resonance energy transfer as well as label-free
impedance measurements that allow monitoring multiple signalling pathways and to assess the structural
determinants of ligand-biased signalling. Using the β1- and β2-adrenergic receptors as study models, we dissected
the signalling cascades engaged by ligands that have biased efficacy toward the adenylyl cyclase, mitogen-
activated protein kinase and calcium pathways and revealed distinct conformational rearrangements of the
signalling modules involved. Combined with molecular modelling of the recently solved 3D structures of the
βARs, these studies should provide the basis for the rational design of drugs with predetermined biased signalling
profiles and improved therapeutic activities.
L-05
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40
Notes

NEW AVENUES TO TARGET WNT/FRIZZLED RECEPTOR
SIGNALING
Guido. J. R. Zaman
Merck Research Laboratories, MSD, Molenstraat 110, 5340 BH, Oss, The Netherlands
Wnt/β-catenin signaling is an important regulator of cell polarity, proliferation and stem cell maintenance during
development and adulthood. Signaling through this pathway is initiated by binding of Wnt proteins to seven-
transmembrane Frizzled receptors, which have structural homology to GPCRs. Aberrant Wnt/β-catenin signaling
has been implicated in a plethora of diseases, most notably colon cancer. We have applied β-galactosidase enzyme
fragment complementation to measure the nuclear translocation of β-catenin, the central transcriptional modulator
in the Wnt/β-catenin pathway. To this end, β-catenin was tagged with a peptide fragment of β-galactosidase and
transfected into cells expressing a corresponding deletion mutant of the enzyme exclusively in the nucleus.
Stimulation of the cells with the Frizzled ligand Wnt-3a increased β-galactosidase activity in a dose-dependent
manner with nanomolar potency. We tested a library of over 2000 synthetic chemical compounds for their ability
to induce or inhibit β-catenin nuclear accumulation. The immunosuppressive protein kinase C inhibitor
sotrastaurin (AEB-071) was identified as an activator of Wnt/β-catenin signaling through its inhibition of glycogen
synthase kinase 3. Furthermore, we identified a class of compounds that activated Wnt/β-catenin signaling with
remarkable cell-specificity and high efficacy. These data show that the β-catenin nuclear accumulation assay can
be used to find new therapeutics targeting the Wnt/β-catenin pathway.
L-06
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Notes

PHARMACOLOGICAL CHARACTERIZATION OF FUNCTIONALLY
SELECTIVE CXCR INHIBITORS
Marcello Allegretti
Dompé S.p.A. Research Center, Via Campo di Pile, snc 67100 – L’Aquila, Italy
The inhibition of CXCL8 is considered as a valid target for the development of innovative treatments for a variety
of severe clinical conditions. The biological activity of CXCL8 is mediated by the interaction with CXCR1 and
CXCR2 membrane receptors belonging to the family of 7TM-GPCRs, expressed on the surface of human
polimorphonuclear cells (PMNs) and of some types of T-cells.
Along the last few years we developed a medicinal chemistry program that led to the characterization of SMW
inhibitors of CXCL8-induced biological activity. Novel classes of potent and selective inhibitors of CXCL8 were
selected and reparixin, the first clinical candidate, is active in the low nanomolar range in inhibition of human
PMN migration induced by CXCL8. Mechanism of action studies have clarified that reparixin acts as a non-
competitive allosteric inhibitor of both CXCL8 receptors, CXCR1 and CXCR2. Further extension of the
MedChem program led to the generation of novel classes of analogues with optimized pharmacokinetic profile
that supported the reliability of the model.
The characterization of the effects of these molecules on signalling led to a more in-depth comprehension of the
“functional selectivity” of this specific class, that is the ability of allosteric modulators to inhibit some of the
effects induced by the endogenous agonist without affecting others. In particular, as chemokine receptors are also
able to scavenge ligands by internalization, the observation that this class of CXCR1/CXCR2 inhibitors does not
interfere with receptor internalization paves the way to a highly selective inhibition strategy that preserves receptor
ability to target the ligand to degradation, possibly avoiding ligand accumulation during a repeated treatment
regimen.
The pharmacological characterization of CXCR1/2 allosteric modulators in I/R injury models has been thoroughly
investigated by preclinical and clinical studies but, in the last period, the availability of the novel molecules has
allowed both the exploration of the therapeutic potential of the class in acute and chronic inflammatory conditions
and the investigation on the physiopathological role of CXCL8 in new therapeutic areas with still high medical
need. In this talk the most recent results from our studies on the pharmacological preclinical characterization of
functionally selective CXCR inhibitors will be discussed.
L-07
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44
Notes

NANOBODIES AS NEW GPCR MODULATORS
Sven Jähnichen 1 , Christophe Blanchetot 2 , Maria Gonzalez-Pajuelo 3 , David Maussang 1 , Ken Y. Chow 1 , Leontien
Bosch 1 , Sindi De Vrieze 3 , Benedikte Serruys 3 , Hans Ulrichts 3 , Wesly Vandevelde 3 , Michael Saunders 3 , Hans J. De
Haard 2,3 , Dominique Schols 4 , Rob Leurs 1 , Peter Vanlandschoot 3 , Theo Verrips 2 , Martine J. Smit 1
1 VU University Amsterdam, The Netherlands. 2 Utrecht University, The Netherlands, 3 Ablynx N.V., Ghent, Belgium, 4 Rega
Institute, Belgium.
G-protein-coupled receptors have so far not been targeted very successfully with conventional monoclonal
antibodies. Here we report the isolation and characterization of the first functional single domain antibodies
(nanobodies) against the chemokine receptor, CXCR4. Two potent nanobodies, were obtained using a time-
efficient whole cell immunization, phage display and counter selection method. The highly selective nanobodies
competitively inhibited the CXCR4-mediated activation of signal transduction cascades and antagonized the
chemo-attractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to
distinct but partially overlapping sites in the extracellular loops. Interestingly, one behaves as a neutral antagonist,
while the other as a partial inverse agonist at the constitutively active CXCR4. Short peptide linkage of the two
nanobodies resulted in significantly increased affinity for CXCR4, full inverse agonism and picomolar activity in
anti-chemotactic assays. Both monovalent and particularly the peptide-linked nanobodies displayed strong anti-
retroviral activity against T cell- and dual-tropic human immunodeficiency virus-1 (HIV-1). Thus, the nanobody
platform is highly effective in generating extremely potent and selective modulators of GPCRs.
L-08
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46
Notes

mGluR4 POSITIVE ALLOSTERIC MODULATOR: NEW PARADIGM
FOR CNS INDICATION TREATMENT
Stephan Schann, 1 Bruno Giethlen, 2 Christophe Morice, 2 Stanislas Mayer, 1 Christel Franchet, 1 Mélanie Frauli, 1
Rachel Rudigier, 2 Delphine Thiarc, 2 Edith Steinberg, 2 Camille G Wermuth, 2 Thierry Langer, 2 Pascal Neuville 1
1 Domain Therapeutics, BioParc, Boulevard Sebastien Brant, F-67400 Illkirch, France
2 Prestwick Chemical, Parc d’innovation, Boulevard Gonthier d’Andernach, F-67400 Illkirch, France
Glutamate, the most commonly occurring neurotransmitter in the brain, is involved in the main neurological
functions either directly or through interactions with other neurotransmitters. In excess, it can also induce cell
death, a phenomenon known as excitotoxicity. Therefore, acting on glutamatergic transmission can reduce
symptoms of neurological diseases but also prevent neuronal cell death.
Glutamate operates through two families of receptors, the ionotropic and the metabotropic glutamate receptors
(respectively iGluRs and mGluRs). This second family and more particularly Allosteric Modulators of mGluRs
are now considered as better therapeutics approach for the treatment of CNS disorders such as schizophrenia,
Parkinson Disease (PD) or anxiety. 1 mGluRs have been divided in three groups according to their sequence
homologies, pharmacological properties and signal transduction pathways. mGluR4, a Gi-coupled group III
mGluR, is mostly presynaptic and has been shown to be involved in regulation of both glutamatergic and
GABAergic transmissions. Its specific localization in the Basal Ganglia, the CNS structure controlling
movements, makes it a highly promising target candidate for the treatment of both symptomatic and
neurodegenerative aspects of PD. 2
Domain Therapeutics and Prestwick Chemical jointly work on the identification and early development of
mGluR4 Positive Allosteric Modulators (PAMs). In the course of this program, new chemical entities were
generated that display nanomolar efficacy for mGluR4, leftward shift of glutamate EC50, increase in glutamate
Emax, subtype selectivity over the other mGluR subtypes and high water solubility. Moreover, these optimized
leads showed efficacies after peripheral administration in two animal models of PD, the reserpine-induced
dyskinesia and the haloperidol-induced catalepsy in mice. This presentation will summarized the scientific
rationale of targeting mGluR4 for PD treatment as well as key aspects of Domain-Prestwick’s programs.
References:
(1) Conn, PJ.; Christopoulos, A.; Lindsley, CW. Nat. Rev. Drug Disco. 2009, 8, 41-54.
(2) Lindsley, CW.; Niswender, CM.; Engers, DW.; Hopkins CR. Curr. Top. Med. Chem. 2009, 9, 949-963.
L-09
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48
Notes

PROSPECTS FOR A TGR5-MEDIATED CONTROL OF GLUCOSE
HOMEOSTASIS: FOCUS ON THE CLINICAL CANDIDATE
S-EMCA (INT-777)
Roberto Pellicciari
Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia, Via del Liceo 1, 06123 Perugia (Italy)
Beyond the regulation of lipid, glucose and their own metabolism by the nuclear receptor FXR, Bile Acids (BAs)
have also been reported to activate TGR5, a GPCR receptor detected in macrophages, gallbladder and intestine,
also present in lower levels in muscle and brown adipose tissue (BAT). Through TGR5, BAs have been shown to
induce energy expenditure by controlling the activity of. Type 2 deiodinase and the consequent activation of the
thyroid hormone in BAT and muscle. TGR5 has also been shown to be expressed in enteroendocrine L-cells
playing a critical role in the control of the incretin glucagon-like peptide-1 (GLP-1) release and in glucose
homeostasis.
We have developed 6-alpha-ethy-23(S)-methyl-cholic acid (EMCA,INT-777), a semisynthetic cholic acid
derivative as a potent TGR5 agonist with no FXR activity and explored its therapeutic properties. Thus, INT-777
was shown to normalize glucose tolerance in obese, insulin resistant (DIO) and diabetic mice, to prevent weight
gain and fat accumulation in mice on a HFD by increasing energy expenditure and fat burning , to induce the
release of GLP-1 in the gut in a TGR5-dependent manner Altogether, the data obtained confirm that TGR5
represents a novel, promising therapeutic target for the treatment of metabolic disorders, and diabesity and
indicate INT-777 as a promising candidate for the treatment of diabesity.
(1) Pellicciari, R.; Gioiello, A.; Macchiarulo, A.; Thomas, C.; Rosatelli, E.; Natalini, B.; Sardella, R.; Pruzanski, M.; Roda, A.;
Pastorini, E.; Schoonjans, K.; Auwerx, J. Med. Chem., 2009, 52(24), 7958-7961. (2) Thomas, C.; Gioiello, A.; Noriega, L.;
Strehle, A.; Outy, J.; Rizzo, G.; Macchiarulo, A.; Yamamoto, H.; Mataki, C.; Pruzanski, M.; Pellicciari, R.; Auwerx, J.;
Schoonjans, K., Cell. Metabolism, 2009, 10(3), 167-177. (3) Macchiarulo, A.; Gioiello, A.; Thomas, C.; Massarotti, A.; Nuti,
R.; Rosatelli, E.; Sabbatini, P.; Schoonjans, K.; Auwerx, J.; Pellicciari, R., J. Chem. Inf. Model., 2008, 48, 1792-180. (4)
Thomas, C.; Pellicciari, R.; Pruzanski, M.; Schoonjans, K.; Auwerx, J., Nat. Rev. Drug Discov., 2008, 7, 678-693.
L-10
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Notes

THE DOPAMINE D1-FAMILY: STEPS TOWARDS SELECTIVITY
Christoph Enzensperger, Dina Robaa, Maria Schulze, Franziska Müller and Jochen Lehmann
Institut für Pharmazie, Lehrstuhl für Pharmazeutische/Medizinische Chemie, Friedrich-Schiller-Universität Jena,
Philosophenweg 14, D-07743 Jena, Germany
The dopamine D1 and the D5 receptor belong to the D1-family, are very similar and at present there are no really
selective compounds available. 1 Relying on the bis-annulated mid-sized azecine-like core we synthesized and
screened a set of ~300 congeners with more or less affinity and selectivity. Here we selected structures (1-4) with
more distinct selectivity further derivatized them in order to improve/work out the selectivity.
H 3 CO
1 2
O
N CH 3
HO
Cl
2 3
CH3 N
H 3CO
N
CH3 3 4
CH3 N
D 1 Affinity [nM]: 35.5 0.83 2.00 3019
D 5 Affinity [nM]: 1.8 0.075 0.23 >10000
D 1 /D 5 Selectivity: 19.7 11 8.7

52
Notes

THE ENDOCANNABINOID PROTEINS AS DRUG TARGETS
Alexandros Makriyannis, Ph.D.
George D. Behrakis Chair in Pharmaceutical Biotechnology
Director, Center for Drug Discovery, Northeastern University
The endocannabinoid biochemical system provides very promising opportunities for therapeutic intervention.
Currently, the CB1 and CB2 cannabinoid receptors, as well as the two critical enzymes involved in
endocannabinoid deactivation, fatty acid amide hydrolase (FAAH) and monoacyglycerol lipase (MGL), are being
seriously explored as therapeutic targets. Our laboratory has been developing biochemical and biophysical
methods to obtain structural and functional information on the interaction of ligands with the target proteins. Such
information is used in the design and development of novel medications. I shall discuss Ligand Assisted Protein
Structure (LAPS), a recently developed approach combining molecular biology and mass spectroscopy-based
focused proteomics. The method is currently being used to study ligand-protein interactions with both GPCRs and
enzymes. Also to be discussed are NMR methods as aids for the design of enzyme inhibitors.
1 (Supported by NIH grants DA3801, DA7215, DA00493)
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Notes

THE ENDOCANNABINOID SYSTEM AND THE REGULATION OF PAIN
AND EMOTION
Daniele Piomelli
Drug Discovery and Development, Italian Institute of Technology, Genoa, Italy
Departments of Pharmacology and Biological Chemistry, University of California, Irvine, CA, USA
The major psychoactive constituent of cannabis, ∆ 9 -tetrahydrocannabinol, influences pain perception and emotions
in humans and laboratory animals by activating brain CB1-type cannabinoid receptors. Two primary endogenous
ligands of these receptors are anandamide, the amide of arachidonic acid with ethanolamine, and 2-
arachidonoylglycerol (2-AG), the ester of arachidonic acid with glycerol. Anandamide and 2-AG are released in
select regions of the brain and are deactivated through a two-step process consisting of transport into cells
followed by intracellular hydrolysis. Selective pharmacological inhibition of anandamide deactivation – by
inhibiting either anandamide transport into cells or its intracellular hydrolysis catalyzed by fatty-acid amide
hydrolase (FAAH) – produces analgesic, anxiolytic-like and antidepressant-like effects in rats. These actions are
not associated with behavioral responses typical of direct-acting cannabinoid agonists and are accompanied by
profound changes in serotonergic adrenergic transmission. On the other hand, selective blockade of intracellular 2-
AG hydrolysis – catalyzed by monoacylglycerol lipase – enhances stress-induced analgesia. These findings
suggest that anandamide and 2-AG contribute to the regulation of pain and emotion, and that the deactivation of
these endocannabinoid lipids might be the target for novel analgesic, anxiolytic and antidepressant drugs.
L-13
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56
Notes

NOVEL POTENT AND SELECTIVE ORL-1 ANTAGONISTS WITH
EFFICACY IN ANIMAL MODELS OF PARKINSON’S DISEASE AND
NEUROPATHIC PAIN
Silvano Ronzoni
NiKem Research srl, Via Zambeletti 25, 20021 Baranzate (MI), Italy.
Since the discovery of the NOP/ORL-1 receptor in 1994, many research groups have devoted a considerable effort
in the identification of selective non-peptide ligands to better elucidate the biological role of this receptor. ORL-1
antagonists in particular have been suggested to be useful, amongst other therapeutic applications, in neuropathic
pain, depression, Parkinson’s disease, food intake inhibition and learning and memory.
During the course of our previous studies we identified a novel ORL-1 antagonist, SB-612111/NiK-10001 (1)
which, notwithstanding a promising preliminary profile (2), was endowed with significant liabilities (mainly high
hERG binding affinity and low oral bioavailability) which hampered its potential as a preclinical candidate. A
medicinal chemistry programme focused at the identification of novel chemical scaffolds was therefore
undertaken, resulting in the discovery of indole derivatives possessing high affinity and selectivity for the ORL-1
receptor.
Detailed structure-activity relationship studies aimed at the optimisation of this new chemical class, with particular
focus on early ADME parameters, will be presented. This investigation led to the identification of NiK-21273, a
potent and selective ORL-1 antagonist with efficacy in animal models of neuropathic pain and Parkinson’s
disease. These results confirmed that ORL-1 antagonists could be of clinical relevance for the treatment of
intractable neuropathic pain and for the symptomatic therapy of parkinsonism.
(1) Zaratin, P.F.et al. J.Pharmacol. Exp. Ther. 2004, 308, 454-461.
(2) Rizzi, A.et al. J.Pharmacol. Exp. Ther. 2007, 321, 968-974.
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Notes

ARE HETEROMERIC G PROTEIN-COUPLED RECEPTORS
RELEVANT TO IN VIVO PHARMACOLOGY?
OPIOID RECEPTORS AS A CASE IN POINT
Philip S. Portoghese
University of Minnesota, Department of Medicinal Chemistry, Minneapolis, MN 55455
Burgeoning evidence for dimers of G protein-coupled receptors (GPCRs) in cultured cells together with the
finding that signaling and trafficking may be modified by such association, raises the likelihood that homomers
and heteromers and may generally be the targets of most drugs that activate or antagonize GPCRs. Until relatively
recently the in vivo relevance of heteromers in the class A GPCR family was not established because both the
assay technology and selective ligands for heteromers were not available. One such class A GPCR family is the
opioid receptors and, in this regard, over a dozen heteromers have been reported in cultured cells. In an effort to
bridge the gap between cultured cells, experimental animals, and humans, we have designed a number of ligands
that potently and selectively activate different heteromeric opioid receptors. Support for the involvement of
heteromers in the action of clinically employed opioid analgesics such as morphine, methadone, fentanyl,
buprenorphine, and nalbuphine has been obtained in culutured cells that contain heteromeric or homomeric opioid
receptors. The finding that some of these clinically employed ligands are devoid of physical dependence while
others produce dependence is related to the specific heteromers that are activated. The screening of ligands on both
heteromeric and homomeric GPCRs in cultured cells offers an approach to develop potent analgesics without
significant side effects.
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60
Notes

STABILIZED GPCRs FOR STRUCTURE BASED DRUG DESIGN
Miles Congreve
Heptares Therapeutics, BioPark, Welwyn Garden City, UK
Heptares has developed a technology that facilitates the study of G-protein-coupled receptors (GPCRs) by
dramatically stabilizing these important proteins outside of the cell membrane. The new stabilized human
receptors (StaRs) are much more robust than the corresponding wild type proteins; they are amenable to
crystallography, biophysical/fragment screening and for raising monoclonal antibodies. The process whereby the
StaRs are first engineered will be explained. The potential of this new technology for structure-based drug design
and biophysical screening of GPCR targets will be presented. The Heptares virtual and fragment screening hit
identification approaches will be outlined with some example results against the A2A receptor target illustrated.
A2A is a validated therapeutic target for treatment of Parkinson’s disease and also of interest for
neurodegeneration. Leads with good ligand efficiency and drug-like properties have been identified from virtual
screening and have been progressed to candidate selection studies in less than 12 months.
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Notes

COMPUTER-ASSISTED LEAD FINDING OF GPCR MODULATORS
Karl-Heinz Baringhaus, Andreas Evers, Clemens Giegerich, Robert Jaeger, Thomas Klabunde
Sanofi-Aventis Deutschland GmbH, CAS Drug Design, Building G 878, 65926 Frankfurt am Main, Germany
Computer-assisted lead finding is a knowledge-driven strategy taking into account public and proprietary chemical
and biological data (e.g. compounds, 3D target structures, biological activities). Building a target family related
chemical and biological space enables lead finding by linking chemotypes to biological targets.
The chemical space of a target family is generated through building databases of biologically active compounds,
privileged fragments, frameworks and pharmacophores. The biological space is comprised through protein
sequences, ligand recognition motifs, 3D structures, 3D pharmacophores and biological activities.
This presentation contains two different methods for GPCR lead finding: 1. All family A GPCRs are described via
three physicochemical properties (Wold scales) of 39 amino acids lining the retinol-like binding site. The resulting
matrix of 39*3 descriptors for each of the 270 GPCRs can be displayed in an in-house tool termed GPCR-
Navigator and gives access to a quantitative similarity description of GPCR pairs. In addition, the tool provides
information on in-house and external GPCR ligands allowing the identification of ligated GPCRs for orphan
receptors as starting point for chemogenomics-based virtual screening. Validation of this GPCR binding site
classification was achieved by a retrospective study revealing its applicability for GPCR lead finding. The
likelihood of success is linked to the GPCR-Navigator defined similarity and this will be exemplified by a recent
successful identification of a nanomolar GPCR agonist. 2. The second approach, termed Pharma 3D provides
sequence-derived 3D-pharmacophore models for GPCRs suitable for virtual screening. This method captures
pharmacophoric features for ligand recognition identified from 13 different GPCR-ligand structures (3 X-ray
structures, 10 homology models). In total, 35 molecular interaction partners (chemoprints) were identified each
encoding the location and identity of a single-feature pharmacophore. This information together with the link to
the corresponding amino acid sequence motif is stored in a pharmacophore building block database. A family A
GPCR can be searched for the presence of such chemoprints in our database and all identified single-feature
pharmacophores are then automatically assembled to a sequence-derived 3D-pharmacophore model. A successful
application of this Pharma 3D approach yielding the discovery of potent agonists of the complement component 3a
receptor 1 (C3AR1) underpins the broad applicability of this concept (1).
(1) Klabunde, T. ; Giegerich, C.; Evers, A. J. Med. Chem. 2009, 52, 2923-2932.
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REGULATION OF 5-HT RECEPTOR FUNCTIONS BY P11
Per Svenningsson 1 , Paul Greengard 2 , Jennifer Warner-Schmidt 2 , Xiaoqun Zhang 1 , Karl Björk 1
1 Karolinska Institutet, Stockholm, Sweden; 2 Rockefeller University, New York, USA
P11 (S100A10) is a member of a large family of S100 proteins that interacts with serotonin receptor 1B
(5-HTR1B) and serotonin receptor 4 (5-HTR4), modulates signal transduction via 5-HT1B and 5-HTR4 receptors
(1, 2). p11 increases 5-HT1B and 5-HTR4 surface expression and facilitates signaling via these receptors. p11 and
5-HTR1B/5-HTR4 mRNAs and proteins are co-expressed in several brain regions. p11 is down-regulated in post-
mortem tissue from depressed individuals, whereas at least two classes of antidepressants (imipramine and
electroconvulsive seizures) increase p11 . P11 knockout mice have a depression-like phenotype and p11 is
required for the behavioral antidepressant responses to 5-HT1B and 5-HTR4 stimulation as well as citalopram and
imipramine in vivo. Likewise, transgenic over-expression of p11 in mice produces antidepressant-like effects in
rodent models of depression. P11 is also involved in L-DOPA-mediated actions in Parkinsonian animals (3).
References
1. Svenningsson P, Chergui K, Rachleff I, Flajolet M, Zhang X, El Yacoubi M, Vaugeois JM, Nomikos GG, Greengard
P. Alterations in 5-HT1B receptor function by p11 in depression-like states. Science. 2006 Jan 6;311(5757):77-80.
2. Warner-Schmidt JL, Flajolet M, Maller A, Chen EY, Qi H, Svenningsson P, Greengard P. Role of p11 in cellular and
behavioral effects of 5-HT4 receptor stimulation. J Neurosci. 2009 Feb 11;29(6):1937-46.
3. Zhang X, Andren PE, Greengard P, Svenningsson P. Evidence for a role of the 5-HT1B receptor and its adaptor
protein, p11, in L-DOPA treatment of an animal model of Parkinsonism. Proc Natl Acad Sci U S A. 2008 Feb
12;105(6):2163-8.
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ALLOSTERIC MODULATION OF NEURONAL NICOTINIC
ACETYLCHOLINE RECEPTORS
Marvin K. Schulte
University of Alaska Fairbanks, Department of Chemistry and Biochemistry, Fairbanks, AK 99775
Nicotinic acetylcholine receptors (nAChR) are involved in mediating acetylcholine based signal transmission in
the CNS and have been implicated in a broad range of CNS disorders including Alzheimer’s disease (1, 2), Autism
(3, 4), Parkinson’s disease (5) and Schizophrenia (6-8). Treatment strategies aimed at increasing activity of
cholinergic systems have until recently focused on acetylcholinesterase inhibitors and partial agonists. Efforts to
develop novel means of modulating nicotinic receptors through allosteric effectors have led to the development of
new classes of nicotinic agents termed positive allosteric modulators (PAMs.) PAMs represent an alternative
treatment strategy since they typically act to enhance responses to endogenous agonist but do not by themselves
elicit responses. Much recent work has focused on α7 nAChR PAMs with a lesser number of studies reporting
compounds that selectively modulate other nAChR subtypes. We have reported the synthesis of a novel type II
PAM selective for the α4β2 nAChR subtype (9, 10). Co-application of dFBr with acetylcholine produces a
biphasic dose response curve with a peak potentiation of over 265% at dFBr concentrations 10 µM. While dFBr does not appear to alter the action of nAChR antagonists, it
is capable of significantly altering the behavior of partial agonists including nicotine. The evaluation of dFBr, its
putative mechanism of action and its potential use as a therapeutic agent in the treatment of disorders involving
nAChR mediated synaptic transmission will be discussed.
(1) Nordberg A. 2001. Biol Psychiatry 49: 200-10
(2) Court J, Martin-Ruiz C, Piggott M, Spurden D, Griffiths M, Perry E. 2001. Biol Psychiatry 49: 175-84
(3) Martin-Ruiz CM, Lee M, Perry RH, Baumann M, Court JA, Perry EK. 2004. Brain Res Mol Brain Res 123:
81-90
(4) Lippiello PM. 2006. Med Hypotheses 66: 985-90
(5) Aubert I, Araujo DM, Cecyre D, Robitaille Y, Gauthier S, Quirion R. 1992. J Neurochem 58: 529-41
(6) Adams CE, Stevens KE. 2007. Front Biosci 12: 4755-72
(7) Friedman JI. 2004. Psychopharmacology (Berl) 174: 45-53
(8) Woodruff-Pak DS, Gould TJ. 2002. Behav Cogn Neurosci Rev 1: 5-20
(9) Sala F, Mulet J, Reddy KP, Bernal JA, Wikman P, et al. 2005. Neurosci Lett 373: 144-9
(10) Kim JS, Padnya A, Weltzin M, Edmonds BW, Schulte MK, Glennon RA. 2007. Bioorg Med Chem Lett 17:
4855-60
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DUALSTERIC GPCR-TARGETING: FINE-TUNING BINDING
SELECTIVITY AND SIGNALLING PATHWAY ACTIVATION
Marco De Amici 1 , Elisabetta Barocelli 2 , Ulrike Holzgrabe 3 , Evi Kostenis 4 , Christian Tränkle 5 , Klaus Mohr 5
1 Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Università degli Studi di Milano, 20133 Milano, Italy;
2 Department of Pharmacological, Biological and Applied Chemical Sciences, University of Parma, Parma 43100, Italy
3 Department of Pharmaceutical Chemistry, Institute of Pharmacy, Julius-Maximilians-University, D-97074 Würzburg,
Germany;
4 Institute of Pharmaceutical Biology and 5 Pharmacology & Toxicology Section, Institute of Pharmacy, Rheinische Friedrich-
Wilhelms-University, D-53121 Bonn, Germany.
An increasing number of G protein-coupled receptors (GPCRs) is recognized to harbor an allosteric binding area
in addition to the orthosteric site for binding of the endogenous messenger substance. If both sites are located in
spatial proximity, ligand molecules should be achievable that bind in an overlap-fashion to both sites
simultaneously. The muscarinic acetylcholine receptor subtypes (M subtypes) are prototypal for the exploration of
allosteric interactions at GPCRs and rather deep insight has been gained in the recent years into the topography of
orthosteric and allosteric ligand binding. To explore the feasibility of the dualsteric (allosteric/orthosteric) design
concept, we fused M2-preferring alkane-bis-ammonio-type allosteric building blocks, which are devoid of
agonistic activity, with orthosteric oxotremorine-like non-selective agonists. Receptor binding and functional
studies revealed M2-preferring agonism of some of the hybrid molecules and proved true dualsteric receptor
binding. Noteworthy, selectivity for the Gi-signaling pathway is achieved by these dualsteric agonists, whereas
conventional orthosteric agonists such as acetylcholine also activate Gs in addition to Gi ((1) and references
therein, (2) for review). Taken together, the design concept of dualsteric agonists is a promising new avenue to
adjust receptor subtype selectivity, efficacy and signaling pathway specificity of GPCR-activating drugs.
(1) Antony, J.; Kellershohn, K.; Mohr-Andrä, M. et al. FASEB J. 2009, 23, 442-450.
(2) Mohr, K.; Tränkle, C.; Kostenis, E.; Barocelli, E.; De Amici, M.; Holzgrabe, U.; Brit. J. Pharmacol., in press.
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POLYPHARMACOLOGY: USING IT TO DIFFERENTIATE AND AS AN
ADVANTAGE WITH SELECTIVE NON-SELECTIVITY
Jonathan S. Mason 1,2
1 Heptares Therapeutics Ltd, BioPark, Broadwater Road, Welwyn Garden City, Herts, AL7 3AX, UK; 2 Lundbeck Research,
Ottiliavej 9, Valby, Copenhagen, DK-2500, Denmark
Polypharmacology is a key concept in the drug discovery process. It is a two-edged sword, where it may be
needed for the effective action of some drugs, but undesirable activities can lead to side effects and toxicity that
limit the use of, or cause the attrition of, an otherwise effective medicine. It does however offer a unique way to
describe and differentiate molecules, based not on the chemical structure or any derived properties, but on how
biological targets see the molecule, by using for example a biological “fingerprint” created by measuring the
binding affinity of a molecule to a large diverse set of relevant biological targets.
In recent years it has become possible to understand much better the extent of polypharmacology present in
medicinal chemistry compounds and the resultant drug candidates. Two major efforts that will be discussed,
highlighting some key findings, are: 1) The Cerep BioPrint® initiative wherein drugs, related reference
compounds, project compounds and attrited compounds (those that failed during development or after launch)
were profiled in a full-matrix manner against 180 pharmacological and ADME targets, with full dose response
measurements for all compounds that had >30% inhibition at 10µM (~4000 compound dataset) and 2) Organized
and integrated databases of structure-activity data (e.g. published, in-house, BioPrint®) that cover much larger
numbers of compounds and targets (e.g. the ChEMBL database has >0.6 million compounds, 7.2 thousand targets
and 2.4 million activities).
Polypharmacology is of particular importance for many GPCR (G-protein-coupled receptor) drugs, and now the
action of ligands can be probed and analyzed at the molecular level following the recent discovery of methods that
enable the stabilization of GPCRs (StaR- Stabilized Receptor technology) and thence two key approaches: 1)
crystallization to yield high resolution X-ray structures with both high and low affinity ligands and 2) biophysical
screening (e.g. Biacore/SPR, TINS/NMR) for kinetics, fragment screening and using site-directed mutants
Biophysical Mapping (Heptares) to probe binding modes/interactions. This stabilization of the receptor is
transformational for GPCR drug research, and enabled the pivotal X-ray structure of the beta-1 adrenergic receptor
with cyanopindolol. The application of the StaR technology platform enables different conformations (agonist,
antagonist…) to be stabilized. Rational approaches to addressing polypharmacology (desired and undesired) are
now possible based upon the target protein binding site and not just ligand-based activity models.
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MULTI-TARGET DRUG DISCOVERY - PAST, PRESENT AND FUTURE
Richard Morphy
Schering-Plough, a part of MSD, Newhouse, Lanarkshire, ML1 5SH. UK.
Currently, drug discovery is largely based upon a “one molecule - one target - one disease” philosophy and many
successful drugs, that are selective for a single target (“magic bullets”), have emerged from this strategy. There is
however an increasing interest in and awareness of multi-target drug discovery (MTDD) within the medicinal
chemistry community as a means of providing a superior therapeutic effect and side effect profile compared to the
modulation of a single target. The inherent redundancy and robustness that exists within biological networks and
pathways means that modulating single proteins may not be sufficient to produce the desired efficacy, whilst at the
same time minimising adverse side effects. It has been demonstrated that a balanced modulation of multiple
targets in parallel can be beneficial for treating diseases with complex etiologies, such as using multi-kinase
inhibitors for treating cancer and multi-target monoaminergic ligands for treating CNS diseases.
Although many currently marketed drugs act via multiple targets, the discovery of their polypharmacological
mechanism of action was usually serendipitous and retrospective. The premeditated design of compounds with a
predefined multi-target profile (i.e. “designed” multiple ligands or DMLs) is a relatively recent development. The
combination of the desirability of discovering multiple ligands on the one hand, and the increasing tractability of a
rational approach on the other, has lead to a strengthening of interest in this area in recent years. Designing
multiple ligands presents medicinal chemists with many challenges and an early assessment of the feasibility of
the approach for a particular target combination is essential. A key objective is to achieve a balanced activity for
each target of interest, whilst obtaining wider selectivity and a suitable oral pharmacokinetic profile.
Historically, there have been two quite different methods of generating lead compounds, screening approaches that
rely largely upon serendipity and knowledge-based approaches that exploit information either from the general
literature or proprietary information from within an organization. New strategies to help medicinal chemists
discover the next generation of DMLs will also be described.
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MULTIPLE MECHANISM DRUGS FOR NEURODEGENERATIVE
DISEASES AND POST-STROKE NEUROPROTECTION
Cornelis J. (Neels) Van der Schyf
Departments of Pharmaceutical Sciences, and Neurobiology, Northeastern Ohio Universities Colleges of Medicine and
Pharmacy, Rootstown, OH, 44313 USA.
Transcriptomics and proteomics profiling suggest that the disease etiologies of neurodegenerative disorders and
post-stroke neurodegeneration are multifactorial in origin. As such, these pathologies offer complex challenges to
drug discovery efforts aimed at preventing or even reversing their progression. In response to this complexity, a
new paradigm has emerged which challenges the widely held assumption that ‘silver bullet’ agents are superior to
‘dirty drugs’ in therapeutic approaches aimed at the prevention or treatment of these diseases. In stroke,
neuroprotective strategies have typically focused on the neurotoxic ischemic environment that threatens neuronal
cells. It has now become clear that the complex processes that occur post-stroke call for the targeting of multiple
pathobiological events (apoptotic and necrotic cell death and inflammatory processes among others) and a
diversity of cells that include glia, vascular epithelial, and inflammatory cells. While the core or umbral area of
ischemic damage in stroke likely cannot be saved from cellular death, the penumbral area surrounding the core,
despite being burdened with severely compromised blood flow, may be preserved either by reperfusion or by the
use of neuroprotective agents. A neuroprotective approach is attractive clinically because, if left untreated, the
penumbral region ultimately will be incorporated in the umbral core, and the lesion will enlarge over time. In this
presentation, I will report on new multimodal drug candidates developed in our laboratory that target dual calcium
entry pathways, i.e. both the L-type calcium channel and the NMDA receptor channel. The effect of one candidate
compound was measured after permanent, and transient (1 h) focal cerebral ischemia in the murine middle cerebral
artery occlusion (MCAO) model. In the permanent MCAO model, infarct area (visualized by TTC staining) was
reduced by 42.6% (P < 0.05) compared to vehicle (DMSO)-treated controls, and brain swelling by 78.3% (P <
0.001), while also in the transient MCAO model, significantly less brain damage (visualized by Fluoro-Jade
staining, 13±7 mm 3 , P < 0.01) was seen in treated animals compared with two control groups (no treatment: 47±4
mm 3 ; vehicle, DMSO: 50±10 mm 3 ). These results, hitherto unexplored drug targets for post-stroke
neuroprotection, and future directions in multiple mechanism drug discovery for neurodegenerative diseases will
be discussed.
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NOVEL THERAPEUTIC APPROACHES CONSTITUTING
MULTIFUNCTIONAL NEUROPROTECTIVE AND
NEURORESTORATIVE DRUGS FOR NEURODEGENERATIVE
DISEASES
Moussa B.H. Youdim
Eve Topf and US National Parkinson Foundations Centers of Excellence for Neurodegenerative Diseases
Research and Teaching Technion-Rappaport Faculty of Medicine, Haifa; Israel and Varinel Inc. and Varinel
LCD (USA-Israel)
Alzheimer’s disease (AD) and Parkinson’s disease (PD), Huntington disease (HD) , amyotrophic lateral sclerosis
(ALS) and multiple Sclerosis (MS) are initiated by cascade of neurotoxic events, that includes oxidative stress,
brain iron dysregulation, glutamate excitotoxicity, nitric oxide, inflammatory process, neurotxic processing
misfolding and aggregation of abeta-peptide, alpha-synuclein resulting from the possible demise of ubiquitin-
proteasome system (UPS) as demonstrated neurochemically and by transcriptomics and proteomic profiling. AD
and PD subjects are benefiting from symptomatic effects of cholinesterase inhibitors, memantine, monoamine
oxidase A and B inhibitors, L-dopa, dopamine receptor agonists, COMT inhibitors and dopamine agonists,
glutame antagonist (memantine),anti viral drug amantadine developed to act on a single molecular target. For HD
and ALS there are no effective drugs. Such drugs have limited symptomatic activities and current
pharmacological approaches have sever limitation in their ability to modify the course of the disease, offering
incomplete and transient benefit to patients. However, the new therapeutic strategies for neurodegenerative
diseases are those in which drug candidates are designed expressly to act on multiple neural and biochemical
targets involved in the neurodegenerative process and to possesses neuropotective and neurorestorative activities.
Thus we have hypothesized and developed a series of innovative novel multifunctional drugs from our successful
neuroprotectiev disease modifying anti Parkinson drug, rasagiline (Azilect, FDA approved 2006), called ladostigil
(phase II clinical studies), M30 and HLA-20. These drugs possess neuroprotective (anti apoptotic) and
neurorestorative activities for AD and PD. There were achieved with the use of multifunctional moieties that
target an array of pathological pathways, each of which is believed to contribute to the cascade that ultimately
leads to neuronal cell death. These moieties include cholinesterase and monoamine oxidase inhibitory activities,
iron chelating , radical scavenging, APP processing to non amyloigenic neuroprotective neurotrophic, soluble
APP alpha, inductions of GDNF, BDNF and HIF (hypoxia inducing factor),VEGF (vascular endothelial growth
factor) and prevention entry into cell cycle via inhibition of G o / G 1 and cycline D. These drugs convert more
than 85% of human embryonic stem cells in to neurons and differentiate several other cells into neurons in
culture. Multifunctional drugs have advantage over the problem of so called monomodal “Magic Bullets” that act
on one neurotoxic modality of neurodegenerative diseases, where there are cascade of neurotoxic event and
pathologies (Youdim and Van Der Schyf, 2009).
Youdim, MBH and Van Der Schyf, C (Editors) (2009) Multifunctional Drugs as Neurotherapeutics, Neurotherapeutics 6; 1-
211.
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CENTRAL OREXIN PEPTIDERGIC SYSTEM AS TARGET FOR NOVEL
TREATMENTS OF INSOMNIA, ANXIETY AND DRUG ADDICTION:
RECENT FINDINGS FROM THE GSK DISCOVERY PERFORMANCE
UNIT
E. Merlo Pich, A. Gozzi, A. Bifone, L. Piccoli, M. Massagrande, P. Martinelli, D Amantini, D. Montanari, S.
Zamuner, P. Bettica, R. Di Fabio, M. Corsi, E. Ratti.
Addiction & Sleep Disorder DPU, Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline Medicines
Research Centre, Verona, Italy.
Orexin/hypocretin neuropeptides (A and B) bind to two G-protein coupled receptors, OX1 and OX2 receptors that
are widely distributed in the mammalian brain with only partially overlapping patterns. A large body of pre-
clinical and clinical evidence suggest that the orexin system is deeply implicated in the sleep disorders, in
particular insomnia, as well as in modulating the reward pathways and anxiety. As part of our internal drug
discovery program aimed towards the identification of potent, selective and bioavailable orexin receptor
antagonists, several preclinical tests and innovative compounds were developed to profile the novel molecules and
to predict possible therapeutic roles. These investigations led to the filing of several patents and assessment of
pre-candidate compounds to identify high quality molecules to be progressed in human studies. The use of some of
these compounds as exploratory tools produced evidence of a differential regulatory role for OX1 receptors vs.
OX2 receptors on behavioural and neurobiologic outcomes. A particular case of translational approach linking
preclinical and clinical data on sleep control will be presented for SB-649868, currently in Phase II, and
GSK1059865, a novel OX1 selective tool compound.
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BIS-AMIDO PIPERIDINE DERIVATIVES AS IN VITRO AND IN VIVO
POTENT DUAL OREXIN RECEPTOR ANTAGONISTS
R. Di Fabio, 1 P. Gerrard, 1 R. Porter, 2 G. Stemp, 2 D. Nash, 2 C. Branch, 2 M. Massagrande, 1 A. Poffe, 1 L. Piccoli, 1
S. Braggio, 1 E. Ratti. 1
1 Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline Medicines Research Centre, Verona, Italy;
2 Neurosciences Centre of Excellence for Drug Discovery, New Frontiers Science Park, Harlow, UK.
The hypothalamic peptides orexin A and orexin B are potent agonists of two G-protein coupled receptors, namely
OX1R and OX2R. These receptors are widely distributed, though differentially, in the rat brain. In particular,
OX1R was found to be highly present throughout the hypothalamus, whilst OX2R was found mainly in the VPN.
A large body of compelling pre-clinical and clinical evidence suggest that the orexin system is deeply implicated
in the sleep disorders. In particular, modulation of the orexin receptors by appropriate antagonists was proven to
be an efficacious strategy for treatment of insomnia in humans.
As part of our internal drug discovery program aimed towards the discovery potent and selective druggable orexin
receptor antagonists, novel bis-amido piperidine derivatives were identified as in vitro potent dual orexin receptor
antagonists showing hypnotic properties in multiple pre-clinical models. The structure-activity relationship of this
new chemical series as well as the profile of the best exemplars selected will be discussed.
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DISCOVERY OF POTENT, DIAZEPANE-CONTAINING DUAL OREXIN
RECEPTOR ANTAGONISTS FOR THE TREATMENT OF INSOMNIA
Anthony J. Roecker 1 ,* Christopher D. Cox 1 , John D. Schreier 1 , Swati P. Mercer 1 , Michael J. Breslin 1 , David B.
Whitman 1 , Jeffrey M. Bergman 1 , Karen M. Brashear 1 , Georgia McGaughey 1 , Michael Bogusky 1 , Scott M. Doran 2 ,
Christopher J. Winrow 2 , Anthony Gotter 2 , Susan L. Garson 2 , Steve V. Fox 2 , Richard L. Kraus 2 , C. Meacham
Harrell 2 , Duane R. Reiss 2 , Kristina Groover 2 , Rodney A. Bednar 4 , Wei Lemaire 4 , Joseph G. Bruno 4 , Kathy L.
Murphy 2 , Chunze Li 3 , Cuyue Tang 3 , Dan Cui 3 , Tamara Cabalu 3 , Thomayant Prueksaritanont 3 , Ken S. Koblan 2 ,
Steven D. Young 1 , George D. Hartman 1 , John J. Renger 2 , and Paul J. Coleman 1
Departments of 1 Medicinal Chemistry, 2 Neuroscience Research, 3 Drug Metabolism, and 4 The West Point Facility for
Automation and Screening Technology (WP FAST), Merck Research Laboratories, WP14-2, P.O. Box 4, Sumneytown Pike,
West Point, Pennsylvania 19486
Orexin antagonists have been identified as possible treatments for a number of
indications including addiction, psychiatric indications, and insomnia. Clinical proof-of-
concept has been achieved using dual orexin receptor antagonists (DORAs) for the treatment of
insomnia. This milestone has piqued interest in orexin antagonist research from academic and
industrial laboratories worldwide. Merck has recently disclosed the discovery and preliminary
optimization of DORAs with a 1,4-diazepane central constraint. This presentation will focus on
the optimization of the metabolic profile and physical properties of this series of compounds
though core and peripheral heterocycle replacements. The in vivo activity of optimized
compounds in mouse and rat sleep experiments will also be discussed.
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OREXIN RECEPTOR ANTAGONISTS – A NEW THERAPEUTIC
PRINCIPLE IN CNS DISORDERS?
Christoph Boss, Ralf Koberstein, Hamed Aissaoui, Thierry Sifferlen, Markus Gude, Romain Siegrist, Jodi
Williams, Christine Brotschi, Martin Bolli, Thomas Weller, Catherine Brisbare-Roch, Francois Jenck, Alexander
Treiber, John Gatfield
Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, CH-4123 Allschwil, Switzerland
Orexins/hypocretins are hypothalamic neuropeptides that play a role in regulating vigilance in mammals, as
suggested by the deficits in orexinergic function that are associated to rodent, canine and human narcolepsy.
However, orexinergic fibers widely distribute in the brain, suggesting that orexins exert broad homeostatic
functions, with a particular role as regulators of behavioral arousal and wake states relating to stress and reward.
Manipulation of the orexin system using brain-penetrant orexin receptor antagonists, or partial, or inverse agonists
may prove therapeutically useful in the treatment of medical and psychiatric conditions associated with disturbed
orexin circuitry in the brain. Selective or dual orexin1-receptor and/or orexin2-receptor ligands (examples given in
Scheme 1 below) that cross the blood-brain-barrier and transiently modify orexinergic function are of key
pharmacological interest for scientific and medical investigations. This presentation summarizes research to
identify and characterize orexin receptor ligands and their biomedical potential for understanding the physiological
and pathophysiological roles of endogenous orexins.
O
O
N
O
N
H
CF3 Almorexant [1]
N
SB-334867
(dual Ox1/Ox2-receptor antagonist;
Clinical Phase 3; Actelion Phramaceuticals Ltd)
[2]
(selective Ox1-receptor antagonist)
N
HN
HN O
Scheme 1: Examples of orexin receptor antagonists with different selectivity profiles.
N
O
Br
Br
N
H
O
N
H
JNJ-10397049 [3]
selective Ox2-receptor antagonist)
[1] Dingemanse, J.; Dorffner, G.; Hajak, G.; Benes, H.; Danker-Hopfe, H.; Polo, O.; Saletu, B.; Barbanoj, M. J.; Pillar, G.;
Penzel, T.; Chiossi, E.; Hoever, P. Proof-of-concept study in primary insomnia patients with almorexant (ACT-078573), a dual
orexin receptor antagonist. Sleep and Biological rhythms 2007, 5, A-194.
[2] Smart, D.; Sabido-David, C.; Brough, S. J.; Jewitt, F.; Johns, A.; Porter, R. A.; Jerman, J. C. SB-334867-A: the first
selective orexin-1 receptor antagonist. Br J Pharmacol 2001, 132, 1179-1182.
[3] Dugovic, C.; Shelton, J.; Sutton, S.; Yun, S.; Li, X.; Dvorak, C.; Carruthers, N.; Atack, J.; Lovenberg, T. Sleep-inducing
effects mediated by selective blockade of orexin OX2 receptors during the light phase in the rat. Sleep 2008, 31, A33.
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O
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NOVEL OXYSTEROLS ACTIVATE THE HEDGEHOG PATHWAY IN
VITRO AND STIMULATE SPINAL FUSION IN VIVO
Wei Xiao 1 , Matthew Epperson 1 , Frank Stappenbeck 1 , Francine Farouz 1 , Mariko Reilly 1 , David Jenkins 1 , Scott
Thies 1 , Farhad Parhami 2 , Jeffrey Wang 3 , Michael Jung 4 , Khanhlinh Nguyen 4
1 Fate Therapeutics, 3535 General Atomics Court, San Diego, CA, 92121
2 Department of Medicine, David Geffen School of Medicine, UCLA, Los Angeles, California, USA
3 Department of Orthopedic Surgery, UCLA, Los Angeles, California, USA
4 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA
Bone morphogenic proteins (BMPs) that induce osteoblast differentiation have been used successfully in humans
in spinal fusion procedures and non-union fracture treatments. However, BMPs are restricted in clinical use due to
safety risks and high costs. Therefore, the identification of small molecules acting as anabolic agents represents a
need for orthopedic medicine, in particular for spinal fusion and fracture repair. In our search for new small
molecule anabolic agents, we found that several naturally occurring oxysterols play a critical role during the
differentiation of pluripotent mesenchymal stem cells to osteoblasts through activation of the Hedgehog signaling 1
pathway. Here, we report on the synthesis and characterization of several novel structural analogs of 20(S)-
hydroxy cholesterol and confirm that these novel oxysterols can induce in vivo osteogenic differentiation of
mesenchymal stem cells in a spinal fusion model.
1. Journal of Biol.Chem., 2008, 282, 12, 8959.
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BRAIN TUMOR STEM CELLS AS A TOOL TO IDENTIFY NOVEL
THERAPEUTICS
Patrizia Tunici and Annette Bakker
Oncology Department, Siena Biotech S.p.A., Strada del Petriccio e Belriguardo, 35. 53100, Siena, Italy
ptunici@sienabiotech.it.
Primary brain tumours often represent the most devastating and difficult to treat tumours. Although enormous
advantages in treating other solid tumours highlighted the last decade, the median survival of glioblastoma
remained the same over the last decades, averaging approximately 1 year. Our paramount inability to successfully
treat brain cancer mostly stems from the lack of full understanding of the underlying brain tumour biology and the
limited success of these strategies is largely due to a high incidence of tumour recurrence after treatment.
Recent evidences suggested that a rare population of stem-like cells (or tumour initiating cells) present in brain
tumours may be responsible for development of highly invasive and chemoresistant recurrence tumours. It is
suggested that not all cells from a given brain tumour have the same ability to proliferate and maintain the growth
of the tumour. Only a relatively small fraction of cells in the tumour, termed BTSC, possess the ability to
proliferate, self-renew extensively and adapt to very different micro-environments. Further investigations have
demonstrated that brain tumour stem-like cells also more closely mirror the phenotype and genotype of the
primary tumors and when injected in vivo they are able to give a tumour that recapitulates the characteristics of the
tumour in the patient. Therefore, it can be suggested that to effectively treat brain tumour, BTSCs need to be
eliminated. If the therapy eliminates only the differentiated cancer cells but spares a significant population of the
BTSCs, the tumour will rapidly reform and the patient will have a clinical relapse.
Since brain tumour stem-like cells (BTSC), share many properties with normal neural stem cells, it was shown that
they can either be isolated from the tumour by making use of certain membrane markers (although which marker
to use is under heavy debate) or can be cultured in stem cell like medium conditions in which they grow as
neurospheres.
In Siena Biotech, we have developed, characterized and validated reliable and reproducible 3D-culture sphere
models derived from human biopsies originating from glioblastoma. We use such them for both target
identification and validation as well as to for in vitro screening purposes and in vivo models.
In the presentation, few application examples of the neurosphere model in neuro-oncology will be presented.
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SUCCESSFUL MODULATION OF TUMOR STEM CELLS WITH
SMALL MOLECULE HEDGEHOG PATHWAY ANTAGONISTS
Russell J. Thomas
Medicinal Chemistry Department, Siena Biotech S.p.A., Strada del Petriccio e Belriguardo, 35, 53100, Siena, Italy
rthomas@sienabiotech.it.
The hedgehog pathway, initially discovered in Drosophila by two Nobel laureates, C Nusslein-Volhard and E
Wieschaus (1), is a major regulator for cell differentiation, tissue polarity and cell proliferation. Studies from many
laboratories reveal activation of this pathway in a variety of human cancers (2). It is thus hoped that targeted
inhibition of hedgehog signaling may be effective in treatment of human cancer.
A number of research and development groups are actively investigating ways of blocking the hedgehog pathway
(3). The most successful approach to date is to antagonize the seven-transmembrane domain receptor Smoothened
(Smo), a key component in transforming hedgehog ligand signaling into growth factor expression.
This presentation describes some of the results achieved in the laboratories of Siena Biotech. The approach has
been to develop ligand-based pharmacophore models using reported antagonists of the Smo receptor and use these
to design new structural classes. This approach, incorporating careful application of physicochemical property
design principles, has led to the identification of a number of new scaffolds with promising levels of activity in a
number of model systems.
(1) Nusslein-Volhard C.; Wieschaus E. Nature, 1980, 287, 795–801.
(2) Yang L., Xie G., Fan Q, Xie J. Oncogene, 2010, 29, 469-481.
(3) Stanton B.Z.; Peng L.F.; Molecular BioSystems, 2010, 6, 44-54.
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THERAPEUTIC MODULATION OF THE WNT SIGNALLING
PATHWAY, AN INNOVATIVE APPROACH FOR ONCOLOGY AND
NEURODEGENERATION
Giovanni Gaviraghi
Siena Biotech S.p.A., Strada del Petriccio e Belriguardo, 35, 53100, Siena, Italy
ggaviraghi@sienabiotech.it.
Inhibition of aberrant Wnt signalling represents an important strategic approach to cancer therapies. A desperate
need for agents able to modulate the pathway is also supported by the lack of chemicals specifically targeting the
Wnt signalling in clinical testing.
The pleiothropic effects of the Wnt pathway activate a broad number of target genes strongly linked to cancer and
involved in all the steps of the disease (insensitivity to anti-growth signals, limitless replicative potential, evasion
of apoptosis, motility and tissue invasion and sustained angiogenesis) in a range of different tumour types;
consequently a pathway-based approach coupled to oncology-relevant phenotypic assays has the potential to
identify inhibitors with different therapeutic applications.
Due to the different mechanisms underlying Wnt pathway activation and the difficulty a priori to identify relevant
therapeutic entry points we decided to focus on a pathway or phenotype screening approach rather than target-
based approaches (e.g. targeting β-catenin or components of the signalling downstream β-catenin). Our strategy is
based on a pathway screening approach with the aim of identifying molecules able to affect different nodes of the
Wnt signalling network. This approach has the advantage of selecting compounds able to interfere with the
signalling pathway and therefore affecting the phenotype and the need of identifying the target later on.
The screening system based on a relevant cellular model (Wnt responsive cell line) with a wild-type genetic
profile (free from mutations in the key Wnt molecules (Axin, APC and β-catenin)) and designed with a “three
step” cascade to suit a primary high-throughput screening campaign delivered different chemotypes (hit series)
with potential different mechanisms of action. The initial hit series selection based on cluster analysis and
chemical structure, physico-chemical properties, biological activity and IP position was further prioritized on the
basis of a minimal SAR (achieved through screening of chemical analogues) and IC50 values. The hit series
prioritized showed in-vitro growth and motility inhibition property in the low micromolar range in different cancer
types. The confidence in the project is also based on the recent discovery of the mechanism of action for one of
our chemical series. We are currently focused on testing the in-vivo activity of the most advanced molecules in
different mouse tumour models.
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THE SUSTAINABILITY OF PHARMACEUTICALS IN THE 21 ST
CENTURY: THE IDLE THOUGHTS OF AN IDLE FELLOW
David J. Triggle
State University of New York, Buffalo, NY USA
“Now, Ladies and Gentlemen, I may perhaps take the liberty of inviting you into the workshop of the
chemotherapist”. So wrote Paul Ehrlich, a considerable optimist writing in 1913 on the eve of WW1 and
mankind’s bloodiest century. Now almost a century later we may consider the progress of drug development and
its future. The capacity to have an enduring flow of pharmaceuticals for the ills of the 21 st century will depend on
science and in particular on the “new biology” based on the genomic discoveries of the late 20 th century, but also
and of equal importance, on science policy – will science endure as part of an independent and self-sustaining
intellectual commons - on how much of the new biology will be private and corporate in nature? - - on economic
policies – is health a public right or a private goods? – what are the effects of the increasing chemical
environmental load on our health? – on health care policies – who and how will the 9.5 billion inhabitants of earth
in 2050 have access to pharmaceuticals and health care? – how will it be paid for? - how will we grapple with the
ethical and moral issues of the new biology – eternal life and happiness or a Pandora’s box of disasters?
References
1. D. J. Triggle, Pharmaceuticals in Society, in, “Green and Sustainable Pharmacy”, Ed. K. Kummerer, Springer,
Berlin, in press, 2010.
2. D. J. Triggle, The chemist as astronaut: searching for biologically useful space in the chemical universe.
Biochemical Pharmacology 78: 217-223, 2009.
3. D. J. Triggle, Treating desires not diseases. Drug Discovery Today, 12: 161-166, 2006.
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SHORT COMMUNICATIONS
97

98
Bloom in “Castelluccio”
“Green Italy” on Monti Sibillini
”

WITHDRAWN
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100
SAR KNOWLEDGEBASE APPROACH FOR EXPLORING TARGET-
LIGAND SELECTIVITY PATTERNS: APPLICATION TO KINASE
TARGET FAMILY
Frédéric Souvay, Ismail Ijjaali, Christelle Morlière, Elodie Dubus, Olivier Barberan
Aureus Pharma, 174 Quai de Jemmapes, 75010 Paris, FRANCE
Protein kinases are important therapeutic targets for today’s drug discovery as they are implicated in many
diseases including cancer and metabolic disorders. AurSCOPE Kinase is one of Aureus Pharma’s knowledgebases
developed to manage chemical and biological knowledge for this target family. Chemical structures of ligands as
well as precise descriptions of their associated targets and all reported in vitro and in vivo pharmacological
responses have been collected from literature (articles and patents). The scope of the present study is to explore the
available structure-activity relationships (SAR) knowledge of kinase target family and identify related target-
selectivity patterns as well as kinase ligands potency and specificity.
S-02

IDENTIFICATION OF GPCR LIGANDS THROUGH DOCKING-BASED
VIRTUAL SCREENING: NOT ONLY CRYSTAL STRUCTURES BUT
ALSO HOMOLOGY MODELS.
Stefano Costanzi 1 , Giulio Ferino 1 , Santiago Vilar 1 , Barkin Berk 1 , Stanislav Engel 2 , Irina G. Tikhonova 1 , T.
Kendall Harden 4 , Kenneth A. Jacobson 3 , Marvin C. Gershengorn 2 , Claudio Cavasotto 5
1 Laboratory of Biological Modeling, 2 Clinical Endocrinology Branch and 3 Molecular Recognition Section, National Institutes
101
of Diabetes and Digestive and Kidney Diseases, NIH, DHHS. 4 Department of Pharmacology, School of Medicine, University of
North Carolina. 5 School of Health Information Sciences, University of Texas Health Science Center at Houston.
Three-dimensional structures of target proteins are remarkably useful tools for the computer-assisted discovery of
novel lead ligands. Due to the paucity of crystal structures of G protein-coupled receptors (GPCRs), the scientific
community has resorted to the construction of homology models which have been applied to the identification of
lead ligands through virtual screening.
Rhodopsin has been for years the only available template. However, recent breakthroughs in GPCR
crystallography have led to the solution of the structures of a few additional receptors. In light of these newly
elucidated crystal structures, we have been able to produce a substantial amount of data to demonstrate that
accurate models of GPCRs in complex with their ligands can indeed be constructed through homology modeling
followed by fully flexible molecular docking. These results have been confirmed by our success in the first blind
assessment of GPCR modeling and docking, organized in coordination with the solution of the X-ray structure of
the adenosine A2A receptor (1).
Using the β2-adrenergic receptor as a case study, we have investigated the applicability of crystal structures and
homology models with different levels of accuracy to the identification of GPCR ligands. In particular, our
controlled docking-based virtual screening experiments have been very successful not only when applied to the
experimental structure, but also with the homology models, yielding a clearly delineated prioritization of a pool of
known binders dispersed within a large set of decoy compounds. Remarkably, the retrieval yields can be further
improved by generating an ensemble of alternative conformations of the receptor that accounts for its flexibility.
These results are very much in line with our actual successes in the identification of lead compounds for several
GPCRs, including the thyrotropin releasing hormone (TRH) receptor, the free fatty acid receptor 1 (FFA1, or
GPR40) and the nucleotide P2Y1 receptor, on the basis of virtual screenings conducted using their homology
models.
(1) Michino M. et al. Nat. Rev. Drug Discov. 2009, 8, 455-463.
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102
FUNCTIONALLY-SELECTIVE SEROTONIN 5-HT2 GPCR DRUGS:
5-HT2C AGONISTS WITH 5-HT2A/2B INVERSE AGONIST ACTIVITY
FOR NEUROPSYCHIATRIC DISORDERS
Raymond G. Booth
Department of Medicinal Chemistry, College of Pharmacy University of Florida, Gainesville, Florida 32610 USA
Serotonin 5-HT2A, 5-HT2B, and 5-HT2C GPCRs signal primarily through Gαq to activate phospholipase C and
formation of inositol phosphates and diacylglycerol second messengers. The human 5-HT2C receptor is found
exclusively in the central nervous system and there is compelling evidence in humans and lab animals that 5-HT2C
receptor activation is pharmacotherapeutic for severe neuropsychiatric disorders such as schizophrenia, as well as,
obsessive-compulsive behavioral disorders such as over-eating and psychostimulant drug abuse. Meanwhile,
5HT2A receptor activation produces hallucinogenic effects and 5-HT2B activation leads to heart valve damage and
pulmonary hypertension, thus, there is zero tolerance for drug activation of these 5-HT2 subtypes. A 5-HT2C
agonist drug that does not also activate 5-HT2A and/or 5-HT2B receptors has not been developed despite lucrative
and beneficial therapeutic impact. Drug discovery targeting the 5-HT2C receptor is challenging because its shares
75% transmembrane sequence identity with 5-HT2A/2B. The highly conserved transmembrane domains and same
second messenger signaling complicates development of ligands that activate 5-HT2C but not 5-HT2A or 5-HT2B
receptors. Our medicinal chemistry program incorporating site-directed mutagenesis and molecular modeling
studies reveals important differences in molecular determinants for activation of the 5-HT2 GPCR family
members. We have developed several novel (4R)-substituted-(2S)-dimethylaminotetralin derivatives that are full-
efficacy agonists at human 5-HT2C receptors, but, inverse agonists at 5-HT2A/2B receptors expressed in clonal cells.
Off-target screening of several analogs indicates no significant affinity at 440 kinases and several-dozen GPCRs,
ion channels, transporters, and signaling/metabolizing enzymes. Accordingly, preclinical studies in laboratory
animals were conducted to assess pharmacotherapeutic activity and safety for 3 lead analogs administered by
intraperitoneal injection. Using 2 different rodent models of schizophrenia, potent and efficacious antipsychotic
activity without weight-gain was demonstrated for 2 analogs. In mice, all 3 analogs demonstrated frank anorexic
activity or no stimulation of food intake at doses that produce antipsychotic effects—no tolerance to the anorexic
effects developed with chronic administration. Inhibition of psychostimulant-induced behavior was demonstrated
for 2 analogs in rats and in vivo potency correlates with in vitro 5HT2A inverse agonist and 5-HT2C agonist
potency. No adverse affects were apparent in the animal studies reported here and detailed toxicology and
pharmacokinetic studies are in progress. Medicinal chemistry, molecular modeling, and in vitro/in vivo
pharmacology data will be presented at the Symposium.
Research Funding: United States National Institutes of Drug Abuse and Mental Health (RO1 DA023928 & MH081193)
S-04

MEDICINAL CHEMISTRY OF NOVEL HISTAMINE H4 RECEPTOR
ANTAGONISTS
Rogier A Smits, Herman D. Lim, Gabriella Coruzzi, Maristella Adami, Enade Istyastono, Erik Haaksma, Obbe P.
Zuiderveld, Bert D. Windhorst, Iwan J. P. de Esch, Rob Leurs
Dept. Medicinal Chemistry, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
103
The histamine H4 receptor is a G protein-coupled receptor that, over the past few years, has been demonstrated to
play an important role in inflammatory and immunomodulatory functions (1). Animal models show great potential
for H4R ligands in the treatment of allergic rhinitis, asthma and pruritis, leading to much interest in this receptor as
a new drug target (2). In our search for new H4R ligands, we took a set of small heterocyclic fragments as a
starting point for a medicinal chemistry campaign. Using ligand based in silico modeling and parallel synthesis
approaches, we developed several chemically distinct ligand series with excellent affinity for the H4R. Several of
the compounds showed inverse agonistic behavior and significant anti-inflammatory properties in vivo in the rat.
(1) Smits, R.; de Esch, I. ; Leurs, R. Drug Disc. Today 2009, 15-16, 745-753.
(2) Thurmond, R. ; Nat. Rev. Drug Discov. 2008, 7:41-53.
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104
NOVEL α1-ADRENORECEPTOR ANTAGONISTS RELATED TO
OPENPHENDIOXAN: BIOLOGICAL EVALUATION
AND α1d COMPUTATIONAL STUDY
Antonio Carrieri, a Alessandro Piergentili, b Fabio Del Bello, b Mario Giannella, b Maria Pigini, b Amedeo Leonardi, c
Francesca Fanelli, d and Wilma Quaglia b
a Dipartimento Farmaco-Chimico, Università di Bari, via Orabona 4, 70125 Bari, Italy
b Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, via S. Agostino 1, 62032 Camerino, Italy
c Recordati S. p. A., Drug Discovery, via Civitali 1, 20148 Milano, Italy
d Istituto Dulbecco Telethon e Dipartimento di Chimica, Università di Modena & Reggio Emilia,
via Campi 183, 41100 Modena, Italy
A series of novel openphendioxan analogues were synthesized and tested at α1-adrenoreceptor (AR) subtypes by
binding and functional assays. The α1d-AR binding profile was also examined by means of 2D, 3D-QSAR together
with docking studies. Multiple regression analysis suggested the relevance of adequate number of heteroatoms in
the whole molecule and of passive membrane diffusion to enhance α1d-affinity. Docking simulations against a
computational structural model of the biological target further proved this evidence and furnished support for
chemiometric analysis, where polar, electrostatic, hydrophobic and shape effects of the ortho substituents in the
phenoxy terminal, most likely governing ligand binding, helped the depiction of pharmacophore hypothesis for the
examined ligands data set.
Thr249
Ile248
Gly247
Asp176
Phe384
Phe365
Phe364
Tyr392
Phe388
Trp361
S-06

PROTEIN KINASES: DOCKING AND HOMOLOGY MODELING
RELIABILITY
Tiziano Tuccinardi, 1,2 Adriano Martinelli 1
1 Department of Pharmaceutical Science, University of Pisa, Italy. 2 Department of Biology, Sbarro Institute for Cancer
Research and Molecular Medicine, Temple University, Philadelphia, Pennsylvania 19122, USA
105
Protein kinases play a major role in eukaryotic signal transduction, via regulation of the phosphorylation states
and, thus, cellular functions of substrate proteins. In order to design highly selective or particular multitarget
kinase ligands, computational chemistry is a very helpful tool for predicting the geometry of kinase-ligand
complexes (by means of docking software) and for providing atomic-resolution models of kinases without an
experimentally known 3D structure (by means of homology modeling techniques).
With regards to the prediction of the geometry of kinase-ligand complexes, is it possible to measure the level of
reliability of docking software to predict the binding pose of ATP binding site ligands (ABSL)?
In order to discuss this question we retrieved from the PDB databank more than 700 X-ray structures of kinases
complexed with ABSL and, using six different docking software (for a total of 17 different docking procedures),
we applied two different level of analysis: a) re-docking of the ligands into their co-crystallized kinases; b)
extended cross-docking studies for all the kinases with more than five reported X-ray complexes.
Re-docking calculations show that there are about sixty percent of possibilities to find a correct ligand pose in the
best docking conditions. The extended cross-docking studies suggest that the docking of an unknown ligand into a
kinase has only 37% of possibilities to find a correct ligand pose. Furthermore a careful analysis of the cross-
docking studies suggests that the docking success is correlated with the binding site similarity and the latter is
strongly affected by the similarity of the ligands. This fact determines that, taking into account only ligands with a
good similarity, there are about 60% of possibilities to find a correct ligand pose even in the cross-docking results.
In literature a large number of kinase 3D structures, developed by means of homology model (HM) techniques, are
reported. Most of them have been obtained using as template the 3D structure possessing the highest sequence
similarity with the target kinase. However, our data suggest that kinases adapt themselves for the interaction with
ABSL and this adaptation appears to be fundamental for the docking prediction. On these bases, we have tested
the hypothesis that better results might be obtained for docking a generic ligand X to a target kinase using a HM of
the target created from a kinase template co-crystallized with a ligand similar to X, than from the crystal structure
of the template with the highest sequence similarity with the target kinase. Our preliminary results suggest that
since the kinase shape has a key role, the use of the template with the highest sequence similarity could not always
be the best choice.
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106
A NEGATIVE ALLOSTERIC MODULATOR OF α7 nAChRs
Małgorzata Dukat, 1 Genevieve S. Alley, 1 Galya R. Abdrakhmanova, 2 Hernán A. Navarro 3
1 Department of Medicinal Chemistry, School of Pharmacy; 2 Department of Pharmacology & Toxicology, School of Medicine;
Virginia Commonwealth University, Richmond, VA 23298, USA; 3 Pharmacology and Toxicology, Discovery Sciences,
Research Triangle Institute, Research Triangle Park, NC 27709, USA
Historically our laboratory was and is interested in understanding and developing SAFiR and SAR for nAChR
(nicotinic acetylcholine receptor) ligands. nAChRs belong to the Cys–loop super family of ligand gated ion
channel receptors (LGICRs). Among them are 5-HT3, GABAA, GABAC, and glycine receptors. nAChRs are the
oldest and most studied receptors among the LGICRs. Neuronal nAChRs form functionally active heteromeric
(e.g., α4β2) and homomeric (e.g., α7) receptors. α7 nAChRs are critical for learning and memory and have been
implicated to play a role in Alzheimer’s disease (AD) and other neurological disorders. Interestingly, α7 nAChR-
selective ligands, either agonist or antagonist, have demonstrated neuroprotective effects against β-amyloid1-42
toxicity associated with AD. One plausible explanation is that these effects may be attributed to rapid and
extensive desensitization of α7 nAChRs as with agonists and inhibition by antagonists.
There is compelling evidence that MD-354 (m-chlorophenylguanidine), an analog developed and synthesized in
our laboratory, when co-administered with (-)nicotine might produce its biological effects through α7 nAChRs.
MD-354 lacks affinity at the major population of neuronal nAChRs (i.e., α4β2) (1). In vivo rodent assays with (-
)nicotine and MD-354, such as drug discrimination, locomotor activity, and thermal nociceptive assays (i.e., hot-
plate and tail-flick) (1) imply that MD-354 might behave as an α7nAChR antagonist. Consequently, MD-354 was
examined for its ability to bind at α7 nAChRs. MD-354 produced < 50% inhibition at a concentration as high as
100 µM. Lacking affinity for α7 nAChRs, a remaining possibility is that MD-354 is a negative allosteric
modulator of α7 nAChRs. Indeed, MD-354 (IC50 = 7.98 µM) was demonstrated to allosterically antagonize the
effect of ACh at α7 nAChRs. Moreover, MD-354 (doses 1 and 10 µM) was inactive at peripheral α3β4 nAChRs
that are associated with undesirable side effects.
Allosteric modulators are attractive but challenging targets in drug design. MD-354 is one of the first negative
allosteric modulators of α7 nAChRs known today.
(1) Dukat M, Wesolowska A, Young S, Bondareva T, Young R, Glennon RA. CPDD 2006 Annual Meeting, Scottsdale,
Arizona. Abstract 202:51.
S-08

CHEMOINFORMATICS APPROACHES FOR ANTI-DEMENTIA
TRADITIONAL CHINESE MEDICINE RESEARCH AND
IMPLICATIONS FOR FINDING POLYPHARMACOLOGY DRUGS
Xue-Juan Li, De-Xin Kong, Hong-Yu Zhang*
College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P R China
107
Dementia, including Alzheimer’s disease (AD), is one of the most threatening diseases to the elderly population.
However, there is no yet efficient therapeutic method to AD. It is of great interest to note that traditional Chinese
medicine (TCM) has accumulated some experiences in the treatment of dementia. TCM is one of the most
potential sources of polypharmacology drugs. TCM featured in its combinatorial use of natural medicines and its
curative effects from integrative contributions of multi-components which especially have advantages in the
treatment of multi-factor and complex diseases, such as dementia. Applying cheminformatics methods to calculate
the similarity of hundreds of components which were identified from most prevalent herbs (Rhizoma Chuanxiong,
Radix Salviae Miltiorrhizae, Radix Polygalae Tenuifoliae, and Rhizoma Acori Tatarinowii) in anti-dementia TCM
formulae through searching traditional Chinese medicine database (TCMD) with drug candidates recorded in
MDL drug data report (MDDR) database. It was found that some components (e.g., tetramethylpyrazine, 3-n-
butylphthalide, miltirone, 9-cis, 12-cis-linoleic acid, baicalin) are associated with anti-dementia and have been
recognized by modern medicine. In addition, Radix Polygalae Tenuifoliae contains 1-hydroxy-3,6,7-trimethoxy
xanthone, which is annotated as an antidiabetic and an aldose reductase inhibitor in MDDR database and thus
helpful to treating diabetes-related cognitive decrements. Therefore, it could be concluded that anti-dementia TCM
formulae indeed contain some modern-medicine-justified anti-dementia components, which are likely to act
synergistically in the fight against dementia. These results have important implications for the rational design of
anti-dementia polypharmacology drugs.
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108
CURCUMIN ANALOGS AS MULTI-TARGET ANTIOXIDANTS: FOCUS
ON MITOCHONDRIA
Elena Simoni, a Michela Rosini, a Christian Bergamini, b Romana Fato, b Maria Laura Bolognesi, a Andrea Cavalli, a,c
Roberto Motterlini, c Giorgio Lenaz, b Carlo Melchiorre. a
a)Department of Pharmaceutical Science, University of Bologna , Via Belmeloro 6, I-40126 Bologna, Italy ; b) Department of
Biochemistry, University of Bologna , Via Irnerio 48, I-40126 Bologna, Italy; c) Department of Drug Discovery and
Development - Italian Institute of Technology, Via Morego 30, I-16163, Genova, Italy
Many lines of evidence suggest that oxidative stress and mitochondria impairment have a central role in age-
related neurodegenerative diseases such as Alzheimer’s disease (AD). In addition, the recent understanding that
mitochondria are at the intersection of the life and death of a cell, particularly through the involvement of
mitochondrial oxidative stress, has made mitochondria a promising target for drug discovery and therapeutic
interventions (1). Thus, considering the oxidative stress as a crucial event on the degenerative cascade, we
designed a series of compounds presenting an antioxidant constrained curcumin-like moiety. Curcumin and its
constrained analogs have currently received remarkable interest as they have a unique conjugated structure which
shows a pleiotropic biological profile. Beside the well-known direct antioxidant activity, curcumin displays a wide
range of properties including anti-inflammatory and anti-amyloidogenic activities. Moreover, curcumin has
recently been demonstrated to be neuroprotective through the induction of the antioxidant enzyme heme
oxygenase-1, showing an additional indirect cytoprotective effect (2).
On the basis of the consideration that mitochondria are a major source of reactive oxygen species and are
particularly vulnerable to oxidative stress, it is plausible that antioxidants that alleviate mitochondrial dysfunction
could be beneficial in AD (3). This prompted us to design mitochondria-targeted antioxidants by connecting
curcumin analogs to different polyamine chains. Polyamines might deliver the corresponding conjugates into the
cell with active polyamine transporters and, acting as lipophilic cations (4), drive the antioxidant moiety
selectively into mitochondria in a membrane potential-dependent manner across the inner membrane. In order to
test if such compounds were preferentially taken up by mitochondria, a fluorescent probe was designed by
combining the phenyl benzothiazole group with the polyamine residue.
(1) Moreira, P. I.; Zhu, X.; Wang, X.; Lee, H.G.; Nunomura, A.; Petersen, R. B.; Perry, G.; Smith, M.A., Biochim. Biophys.
Acta. 2010, 1802, 212-220.
(2) Calabrese, V.; Cornelius, C.; Mancuso, C.; Barone, E.; Calafato, S.; Bates, T.; Rizzarelli, E.; Kostova, AT., Front. Biosci.
2009, 14, 376-397.
(3) Bolognesi, ML.; Matera, R.; Minarini, A.; Rosini, M.; Melchiorre C., Curr. Opin. Chem. Biol. 2009, 13, 1-6.
(4) Murphy, M.P., Biochim. Biophys. Acta. 2008, 1777, 1028-1031.
S-10

IDENTIFICATION OF A NOVEL OX1 ANTAGONIST SERIES BY
HIGH-THROUGHPUT SCREENING AND SDM-DIRECTED
HOMOLOGY MODELING OF OREXIN RECEPTORS
*J. Bentley, *T. Fryatt, *E. Glenn, *D. Hallett, *A. Heifetz, † D. Manikowski, † R. Reifegerste, † X. Wang, † M.
Slack, *D. Vaidya and *M. Whittaker
*Evotec, 114 Milton Park, Abingdon, OX14 4SA, UK
† Evotec AG, Schnackenburgallee 114, 22525 Hamburg, Germany
109
Clinical trials of dual OX1/2 receptor antagonists have shown statistically significant efficacy in increasing sleep
efficiency, total sleep time and percentage of time spent in REM sleep in patients with insomnia. 1 Recent
preclinical findings suggest that a selective OX2 antagonist may be preferred over a dual OX1/2 antagonist. 2
Two high throughput screening campaigns of Evotec’s 250K compound library were conducted against both
orexin receptors utilizing FLIPR-based assays in stably-transfected CHO cells. Molecular target engagement was
confirmed using a non-radioactive GTP binding assay. Cross-species selectivity was evaluated against rat OX1
and rat OX2 receptors. One novel OX1 selective series and two novel OX2 selective series were confirmed. The
novel OX1 selective series will be presented.
Evotec has a developed a hierarchical protocol for GPCR modeling. Initial homology models of OX1 and OX2
GPCR receptors were produced in order to suggest a set of potential key residues for site-directed mutagenesis
(SDM). The SDM study revealed that some of the residues are important to ligand binding and others for receptor
stability. Significant differences in the shift of potency of dual OX1/2 antagonists compared to OX1 selective
antagonists were seen. This additional SDM information was used to refine further the modeling process. Ab-
initio modeling of TM4 and multiple kinks in TM6 of the GPCR model were required due to the significant
differences between Orexin receptors and published 7-TM crystal structures.
(1) Bisbare-Roch, C. et al., Nat. Med., 2007, 13, 150-155.
(2) Dugovik, C. et al., J. Pharmacol. Exp. Ther., 2009, 330(1), 142-151.
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110
SODIUM IONS TOGGLE THE ROTAMER SWITCH IN SODIUM-
SENSITIVE DOPAMINERGIC G-PROTEIN COUPLED RECEPTORS
Jana Selent a , Ferran Sanz b , Manuel Pastor a , and Gianni De Fabritiis c
a Computer-Assisted Drug Design Laboratory, b Integrative Biomedical Informatics Laboratory, c Computational Biochemistry
and Biophysics Laboratory, GRIB, IMIM, Universitat Pompeu Fabra, C/ Dr. Aiguader 88, 08003, Barcelona, Spain
G-protein coupled receptors, the largest family of proteins in the human genome, are involved in many signaling
pathways regulated by orthosteric ligand binding and allosteric modulation. Dopaminergic receptors which belong
to the class A family of G-protein coupled receptors take part in the control of various neurological processes and
are therefore important drug targets in the treatment of central nervous system disorders (e.g. schizophrenia,
Parkinson disease). Recent studies indicate that sodium ions allosterically modulate the dopaminergic D2 receptors
affecting mainly agonist binding at the orthosteric binding site (1, 2). In an effort to understand the mechanisms of
allosteric modulation of sodium ions for the dopaminergic D2 receptor, we performed microsecond scale all-atom
molecular dynamics simulations to characterize sodium binding and modulation. We found that sodium ions enter
the receptor from the extracellular side and bind at the residue Asp2.50, an allosteric site deep inside the receptor.
Most importantly, the presence of a sodium ion at this allosteric site provokes a conformational change of the
rotamer toggle switch Trp6.48 which locks in a state identical to the inactive state of the crystallized human β2
adrenergic receptor (3). This study provides novel insights into the effect of sodium ions on receptor conformation
and its importance for the allosteric modulation of G-protein coupled receptors.
(1) Ericksen, S.S.; Cummings D.F.; Weinstein H.; Schetz J.A. J. Pharmacol. Exp. Ther. 2009, 328, 40-54.
(2) Schetz J.A. Mini. Rev. Med. Chem. 2005, 5, 555-61.
(3) Selent J., Sanz F.; Pastor M.; De Fabritiis G. 2010, (submitted).
S-12

POSTER SESSION
111

112
Pizzo del Diavolo
Monte Bove

MOLECULAR MODELING ANALYSIS OF THE INTERACTIONS OF
THE HUMAN ADENOSINE A2A RECEPTOR WITH ITS LIGANDS
Barkin Berk 1 , Santiago Vilar Varela 1 , Kenneth A. Jacobson 2 , Stefano Costanzi 1
1 Laboratory of Biological Modeling and 2 Molecular Recognition Section, National Institutes of Diabetes and Digestive and
Kidney Diseases, NIH, DHHS,USA.
113
In 2008, the human adenosine A2A receptor and its moderately selective, high-affinity antagonist ligand,
ZM241385 (4-2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethylphenol) joined the
shortlist of GPCRs for which an X-ray crystallographic structure has been determined (1). Moreover, over the
course of the years, many studies and reviews were published concerning its homology modeling, structure of
binding site, various synthesis of agonist /antagonists and their structure and activity relationships (2-3). All these
data indicate that typical antagonists and agonists of A2A AR bind with a mode similar to the one shown by
ZM241385, forming a key H-bond with Asn253 in TM6 with their exocyclic NH groups. Additionally, published
docking studies suggest that the 2′- and 3′-OH groups of the ribose moiety, essential for agonistic activity, bind in
proximity of Ser277 and His278 and are putatively involved in H-bonding interactions with these residues.
To further elucidate the modes of binding of ligands of the human A2A adenosine receptor and to establish the key
differences between agonists and antagonists, in this study more than 200 ligands based on various chemical
scaffolds were selected from many publications and subjected to molecular docking. The docking was performed
using the bicyclic core of ZM241385 as a constraint and various criteria for the selection of the poses.
The “protein-ligand interaction fingerprints” and “protein-ligand interaction fields” as implemented in the
Molecular Operating Environment (MOE) were also used as tools to highlight the role of the various amino acids
in the interaction with the ligands.
(1) Jaakola VP. et al. Science. 2008 , 322, 1211–1217.
(2) Cristalli G. et al. ChemMedChem. 2007, 2( 3), 260-281.
(3) Ivanov AA. et al. J Med Chem. 2009, 52 (10), 3284-3292.
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EFFECTS OF 5'-CHLORO-5'-DEOXY-N 6 -(±)-ENDO-NORBORNYL-
ADENOSINE, A POTENT AND HIGHLY SELECTIVE A1 ADENOSINE
RECEPTOR AGONIST, ON NEUROPATHIC PAIN-INDUCED
BEHAVIOURAL AND MORPHOLOGICAL CHANGES IN SPINAL
MICROGLIA
Livio Luongo, a Loredana Cappellacci, b Catia Giordano, a Riccardo Petrelli, b Francesca Guida, a Palmarisa
Franchetti, b Mario Grifantini b and Sabatino Maione a
Department of Experimental Medicine, The Second University of Naples, via Costantinopoli 16, 80138 Naples.
School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, via S. Agostino 1, 62032 Camerino
This study was undertaken in order to investigate the effect of chronic treatment with 5'-chloro-5'-deoxy-N 6 -(±)-
endo-norbornyladenosine (5'Cl5'd-(±)-ENBA), 1 a potent and highly selective adenosine A1 receptor agonist, on
thermal hyperalgesia and mechanical allodynia in a mouse model of neuropathic pain, the spared nerve injury
(SNI). Chronic systemic administrations of 5'Cl5'd-(±)-ENBA (0.5 mg/kg, i.p once a day) reduced both thermal
hyperalgesia and mechanical allodynia 3 and 7 days after SNI, in a way prevented by DPCPX (3 mg/kg, i.p.), a
selective A1 receptor antagonist. SNI induced spinal changes on microglial activation ipsilaterally to the nerve
injury. Moreover, 5'Cl5'd-(±)-ENBA significantly reduced microglial activation in vitro. In particular pre-
incubation with 5'Cl5'd-(±)-ENBA prevented the microglial morphological changes induced by LPS, ATP,
LPS+ATP challenges. Our results demonstrated an involvement of A1 receptors in the increase of nociceptive
thresholds and in spinal changes occurred in neuropathic pain. In addition, 5'Cl5'd-(±)-ENBA antiallodynic and
antihyperalgesic effects could be mediated by the A1 receptors expressed on microglial cells.
(1) Franchetti, P.; Cappellacci, L.; Vita, P.; Petrelli, R.; Lavecchia, A.; Kachler, S.; Klotz, K.-N.; Marabese, I.; Luongo, L.;
Maione, S.; Grifantini, M. J. Med. Chem. 2009, 52, 2393-2406.
P-02

MOLECULAR MODELING STUDY ON POTENT AND SELECTIVE
ADENOSINE A3 RECEPTOR AGONISTS
Diego Dal Ben, Michela Buccioni, Dania Giacobbe, Catia Lambertucci, Gabriella Marucci, Deepak Shankar
Panmand, Rosaria Volpini, Gloria Cristalli
School of Pharmacy, Medicinal Chemistry Unit, University of Camerino; Via S. Agostino 1, 62032 Camerino (MC), Italy
115
Adenosine A3 receptor (AA3R) is involved in a variety of key physiological processes such as release of
inflammatory mediators and inhibition of tumor necrosis factor-α production. Its activation is suggested to take
part in immunosuppression and in the protection from brain and heart ischemia. Recent studies suggest that AA3R
agonists could be employed as therapeutic agents for the treatment of rheumatoid arthritis, dry eye disorders,
asthma, as anti-inflammatory agents, and in cancer therapy as cytostatic and chemoprotective compounds (1).
Hence, the design and synthesis of potent and selective AA3R agonists could help to provide tools for further
characterization of the physiopathological role of this receptor and for the development of new drugs. Previously
reported 2-(ar)alkynyl-N 6 -methoxyadenosine and corresponding MECA (5’-N-methylcarboxamidoadenosine)
derivatives demonstrated to possess different degrees of affinity and selectivity for the human AA3R (2). Basing
on further previous studies demonstrating that also the introduction of a methyl group in N 6 -position of 2-
phenylethynyladenosine favoured the interaction with the human AA3R (3), we designed and synthesised 2-
aralkynyl-N 6 -methylMECA analogues with the aim at improving AA3R affinity and selectivity. Biological
evaluation of the compounds proved that the new nucleosides behave as full AA3R agonists, possessing sub-
nanomolar AA3R affinity and remarkable selectivity versus the other adenosine receptor (AR) subtypes, hence
resulting to be among the most potent and selective AA3R ligands reported so far (4). In this study, we made an
attempt to get a rationalization of the high affinities and selectivities of these molecules for the human AA3R, by
using AR structural models based on the AA2AR crystal structure and molecular docking analysis. Post-docking
analysis allowed to assess the ability of modeling tools in predicting AA3R affinity and in evaluating the influence
of substituents on affinity and selectivity.
References:
(1) Cristalli, G.; Volpini, R. Adenosine Receptors: Curr. Top. Med. Chem. 2003, 3, 355-469.
(2) Volpini, R.; Dal Ben, D.; Lambertucci, C.; Taffi, S.; Vittori, S.; Klotz, K.-N.; Cristalli, G. J. Med. Chem. 2007, 50, 1222-
1230.
(3) Volpini, R.; Costanzi, S.; Lambertucci, C.; Taffi, S.; Vittori, S.; Klotz, K.-N.; Cristalli, G. J. Med. Chem. 2002, 45, 3271-
3279.
(4) Volpini, R.; Buccioni, M.; Dal Ben, D.; Lambertucci, C.; Lammi, C.; Marucci, G.; Ramadori, A. T.; Klotz, K.-N.; Cristalli,
G. J. Med. Chem. 2009, 52, 7897-7900.
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116
SYNTHESIS AND BIOLOGICAL EVALUATUION OF POTENT
LIGANDS FOR THE PUTATIVE GUANOSINE RECEPTOR
Ajiroghene Thomas, Michela Buccioni, Diego Dal Ben, Catia Lambertucci, Carmen Lammi, Gabriella Marucci,
Rosaria Volpini, Gloria Cristalli
School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, via S. Agostino, 1, 62032 Camerino, Italy.
A variety of cells releases guanosine and its nucleotides under physiological and pathological conditions. The
amount of guanine-based purines released from astrocytes is fivefold much greater than that of their adenine-based
counterparts (1). There are extensive evidences that extracellular guanosine exerts a plethora of biological effects
including trophic modulations in the nervous system, promotion of cell proliferation and differentiation,
neuroprotective actions, inhibition of glutamatergic transmission, and antiapoptotic effects (2).
Specific binding sites for [ 3 H]-guanosine, which are not recognized by other purinergic receptor ligans, were
detected on membrane preparations from rat brain (3). These findings supported the hypothesis of the existence of
a specific G protein-coupled receptor (GPCR) for guanosine.
On this basis, various ligands were designed and synthesized for the further characterization of this elusive binding
site. In particular, the synthesis of new ligands involved modifications of guanosine at the 6- and 5-positions.
These molecules were tested through an innovative Eu-GTP binding assay, which, by using the time-resolved
fluorescence technique, allows to assess the coupling of a receptor to a G protein.
The tested compounds seems to activate a GPCR with different potencies, in some case even higher than that of
guanosine itself.
References:
(1) Ciccarelli, R.; Di Iorio, P.; Giuliani, P.; D’Alimonte, I; Ballerini, P.; Caciagli, F.; Rathbone, M.P. Glia 1999, 25, 1, 93-98.
(2) Traversa, U.; Bombi, G.; Di Iorio, P.; Ciccarelli, R.; Werstiuk, E. S.; Rathbone, M. P. Br. J. Pharmacol. 2002, 135, 4, 969-
976.
(3) Traversa, U.; Bombi, G.; Camaioni, E.; Macchiarulo, A.; Costantino, G.; Palmieri, C.; Caciagli, F.; Pellicciari, R. Bioorg.
Med. Chem. 2003, 11, 24, 5417-5425.
P-04

SYNTHESIS OF NOVEL LIGANDS FOR P2X3 RECEPTOR
Deepak Shankar Panmand, 1 Catia Lambertucci, 1 Diego Dal Ben, 1 Gabriella Marucci, 1 Rosaria Volpini, 1
Andrea Nistri, 2 Gloria Cristalli 1
1 School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, I-62032 Camerino, Italy; 2 Sector of Neurobiology
and Italian Institute of Technology Unit, International School for Advanced Studies (SISSA), Via Beirut 2/4, 34014 Trieste,
Italy
117
Purinergic receptors are involved in various physiological and pathological conditions like brain trauma ischemia,
and neurodegenerative diseases. They are classified into P1 and P2 receptors activated by adenosine and several
nucleotides, respectively. Among P2 receptors the P2X subtypes are ligand gated ion channels while P2Y subtypes
are G-protein coupled receptors. P2X3 subtype is activated by adenosine-5’-triphosphate (ATP) and it plays an
important role in neuropathic and chronic pain pathways. Hence, ligands able to block these receptors could be
useful for the treatment of chronic pain conditions and migraine (1).
Recently, a number of purine acyclic-nucleotides have been synthesized and proved to behave as partial agonists
of P2X3 receptors (2). Furthermore, since non-nucleotide diammino-pyrimidine derivatives were reported to block
P2X3 receptors with high potency (3), a new series of P2X3 ligands was designed taking into account the structure
of the acyclic nucleotides and molecular modeling data obtained by comparing pyrimidine and purine scaffolds.
Hence, purine derivatives bearing a substituted benzyl chain in 9-position were synthesized and evaluated on
HEK293 cells expressing rat P2X3 receptors using patch clamp technique.
Results demonstrated that the new compounds behave as P2X3 receptor antagonists, endowed with IC50 activity in
the micromolar range.
References:
(1) Burnstock G. Pharmacol. Rev. 2006, 58, 58–86.
(2) Volpini, R.; Mishra, R. C.; Kachare, D. D.; Dal Ben, D.; Lambertucci, C.; Antonini, I.; Vittori, S.; Marucci, G.; Sokolova,
E.; Nistri, A.; Cristalli, G. J. Med. Chem. 2009, 52, 4596–4603.
(3) Carter, D. S.; Alam, M.; Cai, H.; Dillon, M. P.; Ford, A. P. D. W.; Gever, J. R.; Jahangir, A.; Lin, C.; Moore, A. G.;
Wagner, P. J.; Zhai, Y. Bioorg. Med. Chem. Lett. 2009, 19, 1628-1631.
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118
ALLOSTERIC ACTIVITY OF POTENT CIRAZOLINE ANALOGUES
TOWARDS ADRENERGIC AND IMIDAZOLINE RECEPTORS
Marialessandra Contino, a Nicola A. Colabufo, a Roberto Perrone, a Maria Pigini, b Francesca Ghelfi, b Russell
Thomas c and Antonio Carrieri a
a Dipartimento Farmaco-Chimico, Università di Bari, via Orabona 4, 70125 Bari, Italy
b Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, via S. Agostino 1, 62032 Camerino, Italy
c Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy
Imidazoline receptors (I2R) are involved in the modulation of some behavioral states such as depression, anxiety
and in vivo alterated receptor density indicate their role in the modulation of catecholamines release in the CNS
(1). The presence of these receptors has been also demonstrated in gastric and intestinal tissues. Imidazoline-like
structures, such as cirazoline, have been characterized as modulators of cholinergic motor function in guinea-pig
ileum by a direct interaction with presynaptic α2D-adrenoceptors while an involvement of presynaptic imidazoline
receptors was not reported (2). Moreover, an involvement of α1-adrenoceptors in mediating relaxation of
carbachol-evoked contraction in rat ileum has been showed (3) and an interaction between α1 adrenoceptors
activation and I2R in the same organ has been hypothesized (4). Cirazoline and some structurally related
compounds, previously characterized as potent α2-adrenoceptor ligands (5), are here studied in isolated guinea pig
ileum for determining I2, α1 post-junctional and α2 pre-junctional activities, and their pharmacological profiles,
endowing different (ant)agonsim together with allosterism, are presented.
X
O
R
NH
N
R X
Cirazoline H cyclopropyl
1 CH3 m-NO2-C6H4
2 CH3 C6H5
(1) Nutt, D. J.; French, N.; Handley, S.; Hudson, A.; Husbands, S.; Jackson, H.; Jordan, S.; Lalies, M. D.; Lewis, J.; Lione, L.;
Mallard, N.; Pratt, J. In The Imidazoline Receptor. Pharmacology, Functions, Ligands, and Relevance to Biology and
Medicine; Reis, D.J.; Bousquet, P.; Parini, A.; Eds.; The New York Academy of Sciences: New York, 1995, Vol. 763, 125-139.
(2) Colucci, R.; Blandizzi, C.; Carignani, D.; Placanica, G.; Lazzieri, G.; Del Tacca, M. Effects of imidazoline derivatives on
cholinergic motility in guineapig ileum: involvement of presynaptic alpha2-adrenoceptors or imidazoline receptors? Naunyn-
Schmiedeberg’s Arch. Phrmacol. 1998, 357, 682-691.
(3) Liu, L.; Coupar, I. M. Characterisation of pre- and postsynaptic-adrenoceptors in a modulation of rat ileum longitudinal and
circular muscle activities. Naunyn Schmiedeberg’s Arch Pharmacol. 1997, 356, 248-225.
(4) MacInnes, N.; Handley, S. L. Potential serotonergic and noradrenergic involvement in the discriminative stimulus effects of
the selective imidazoline I 2-site ligand 2-BFI. Pharmacol. Biochem. Behav. 2003, 73, 427-433.
(5) Crassous, P. A.; Cardinaletti, C.; Carrieri, A.; Bruni, B.; Di Vaira, M.; Gentili, F.; Ghelfi, F.; Giannella, M.; Paris, H.;
Piergentili, A.; Quaglia, W.; Schaak, S.; Vesprini, C.; Pigini, M. α 2-adrenoreceptors profile modulation. 3. (R)-(+)-mnitrobiphenyline,
a new efficient and α 2C-subtype selective agonist. J. Med. Chem. 2007, 50, 3964-3968.
P-06

ENANTIOSELECTIVE INTERACTIONS TO IMPROVE ADRENERGIC
α2C-AGONISM/α2A-ANTAGONISM
Fabio Del Bello, a Laura Mattioli, a Aniket Farande, a Francesca Ghelfi, a Mario Giannella, a Alessandro Piergentili, a
Wilma Quaglia, a Marina Perfumi, a Russell Thomas, b Carla Marchioro, c Michele Dal Cin, c Maria Pigini a
119
a Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, via S. Agostino, 1, 62032 Camerino, Italy
b Siena Biotech S.p.A., Strada del Petriccio e Belriguardo, 35, 53100 Siena, Italy
c GlaxoSmithKline Medicine Research Center, via Fleming, 4, 37135 Verona, Italy
Recently (1) we demonstrated that compounds 1 and 2 behaved as potent and selective α2C-AR agonists and
effective α2A-AR antagonists. It is known that in clinical pain management, α2C selective agonists devoid of the
sedative side effects associated with α2A-AR stimulation might represent alone or in combination with opioid
analgesics an improvement over current therapies with clonidine-like drugs. Therefore, 1 and 2 co-administered
with morphine were tested using the mouse tail-flick test. A very low dose (0.05 mg/kg) of both compounds
caused a significant increase in morphine analgesia comparable to that obtained with a 0.5 mg/kg dose of
clonidine. In addition, 1 and 2 were devoid of sedative side effects. This promising behaviour is being
strengthened by the observation that 1 and 2 are endowed with good chemical stability and exhibit high passive
diffusion through an artificial phospholipid membrane (PAMPA), which could permit them to interact with their
target at a non-surface exposed binding site. The presence of a stereocentre in 1 and 2 prompted us to investigate
the stereochemical requirements for a possible α2C-AR agonism/α2A-AR antagonism improvement. Therefore, the
novel R and S enantiomers of 1 and 2 were prepared and their biological profiles were determined by binding and
functional assays. Moreover, their effects on morphine analgesia modulation were evaluated. In addition, for
racemic compound 1 and its corresponding enantiomers (R)-(-)-1 and (S)-(+)-1 the study was also extended to the
acquisition and expression of morphine tolerance and dependence.
(±)-1
(S)-(+)-1
(R)-(−)-1
(1) Cardinaletti, C.; Mattioli, L.; Ghelfi, F.; Del Bello, F.; Giannella, M.; Buzzone, A.; Paris, H.; Perfumi, M.;
Piergentili, A.; Quaglia, W.; Pigini, M. J. Med. Chem. 2009, 52, 7319-7322.
P-07
(±)-2
(S)-(+)-2
(R)-(−)-2

120
NOVEL MUSCARINIC ANTAGONISTS DESIGNED ON THE 1,4-
DIOXANE SCAFFOLD
Fabio Del Bello, a Aniket Farande, a Yogita Farande, a Mario Giannella, a Maria Pigini, a Alessandro Piergentili, a
Wilma Quaglia, a Marta Nesi, b Rosanna Matucci b
a Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, Via S. Agostino 1, 62032 Camerino, Italy
b Dipartimento di Farmacologia, Università di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy
The five subtypes of muscarinic receptors (M1-M5) are widely distributed in multiple organs and tissues and are
critical to the maintenance of central and peripheral cholinergic neurotrasmission. Smooth muscle expresses
several muscarinic receptor subtypes, the most important being M2 and M3 subtypes from a functional point of
view. Although the M2 muscarinic subtypes are predominant, the smaller population of M3 subtypes appears to be
the most functionally important for treating various medical conditions associated with improper smooth muscle
function, such as overactive bladder (OAB), chronic obstructive pulmonary disease (COPD), and pain-
predominant irritable bowel syndrome (IBS). Due to the wide distribution of muscarinic receptors in the body,
significant systemic exposure to muscarinic antagonists is associated with effects such as dry mouth, constipation,
mydriasis, urinary retention and tachycardia. Therefore, the discovery of novel muscarinic antagonists showing
target organ selectivity would be very useful (1).
The (1,4-dioxan-4-yl)-N,N,N-trimethylmethanaminium iodide nucleus has proved to be a suitable scaffold for its
ability to give potent muscarinic agonists (1) or antagonists (2), depending on the size of the substituent in position
6 (methyl or bulkier groups, respectively) (2). Therefore, we designed, prepared and studied, at all five muscarinic
receptor subtypes, analogs obtained by replacing the hydrophobic portion in position 6 of 2 with groups of
different nature and bulk. The pharmacological profile of the novel compounds was evaluated by receptor binding
assays on chinese hamster ovary (CHO) cells, expressing the five human muscarinic receptor subtypes (hM1-hM5).
From preliminary results, compound 3 showed at all subtypes nM affinity values, which are comparable to those
of some compounds used in therapy for the treatment of OAB.
H 3C
O
O
(1) Peretto, I.; Petrillo, P.; Imbimbo, B. P. Med. Res. Rev. 2009, 29, 867-902.
(2) Piergentili, A.; Quaglia, W.; Giannella, M.; Del Bello, F.; Bruni, B.; Buccioni, M.; Carrieri, A.; Ciattini, S. Bioorg. Med.
Chem. 2007, 15, 886-896.
N + (CH 3) 3 I -
1 2
O
O
P-08
N + (CH 3) 3 I -
O
O
3
N + (CH 3) 3 I -

SYNTHESIS, BIOLOGICAL EVALUATION AND DOCKING STUDIES
OF NR2B/NMDA RECEPTOR ANTAGONISTS
Rosaria Gitto, a Laura De Luca, a Stefania Ferro, a Maria Rosa Buemi, a Francesca Scicchitano, b Giovambattista De
Sarro, b Lara Costa, c Lucia Ciranna c and Alba Chimirri a
a Dipartimento Farmaco-Chimico, Università di Messina, Viale Annunziata, 98168 Messina, Italy
b Dipartimento di Medicina Sperimentale e Clinica, Università Magna Græcia, Germaneto, 88100 Catanzaro, Italy
c Dipartimento di Scienze Fisiologiche, Università di Catania, Viale Andrea Doria 6, 95125 Catania, Italy.
121
NMDA receptors (NMDARs) are involved in many physiological processes, such as neuronal development,
learning and memory but also play important roles in pathological states of CNS, including strokes, seizures, and
pain. NMDARs are receptor complexes containing NR1 and NR2 subunits and, more rarely, NR3 subunit. NR2
subunits exist as four subtypes, each encoded by a distinct gene and named NR2A-D. It is well-known that the
noncompetitive NR2B-antagonists, such as ifenprodil (1) and Ro 25-6981 (2), have good potential as
neuroprotective agents and have been shown to bind at the so-called NR2B ifenprodil site. In previous studies we
reported a combined ligand- and target-based approach which led to the identification of new selective ligands
containing an indole scaffold (1-2). The most active compounds (e.g. 3) proved to prevent audiogenic seizures in
DBA/2 mice and reduce NMDA receptor-mediated current in patch clamp experiments.
N
OH
OH
1, ifenprodil 2, Ro 25-6981
N
OH
OH
R
O
N
H
3, R = H, OMe, OH
In attempt to obtain new insights into ifenprodil-like ligands we synthesized new indole derivatives and screened
their receptor affinity. We also tested the anticonvulsant properties in DBA/2 mice and evaluated the functional
activity by electrophysiological experiments. Moreover, docking experiments were carried out to rationalize the
obtained biological results.
(1) Gitto, R.; De Luca, L.; Ferro, S.; Occhiuto, F.; Samperi, S.; De Sarro, G.; Russo, E.; Ciranna, L.; Costa, L. Chimirri, A.
ChemMedChem 2008, 3, 1539-1548.
(2) Gitto, R.; De Luca, L.; Ferro, S.; Citraro, R.; De Sarro, G.; Costa, L.; Ciranna, L. Chimirri, A. Bioorg. Med. Chem. 2009,
17, 1640-1647.
Research supported by MiUR (PRIN 2008, Grant No 20085HR5JK_002)
P-09
N

122
SYNTHESIS OF NEUROPEPTIDE S RECEPTOR LIGANDS
Dania Giacobbe, 1 Diego Dal Ben, 1 Catia Lambertucci, 1 Gabriella Marucci, 1 Girolamo Calò, 2 Rosaria Volpini, 1
Gloria Cristalli 1
1 School of Pharmacy, Medicinal Chemistry Unit, University of Camerino Via S. Agostino, 62032 Camerino, Italy; 2
Department of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, via Fossato di Mortara,
44100 Ferrara, Italy
Neuropeptide S (NPS) is a recently identified transmitter constituted by 20 amino acids and it is involved in the
modulation of anxiety, arousal, food intake, and insomnia. NPS activates a recently dehorphanized G protein-
coupled receptor (NPSR) and the NPSR is distributed in many brain areas with high expression levels in cortex,
hypothalamus, amygdala and multiple midline thalamic nuclei. Many of these areas have been functionally
associated with arousal and processing of emotional behaviour and, on this basis, NPSR ligands could have
therapeutic potential (1).
Up to date, limited information is available about nonpeptide NPSR ligands, as only one series of bicyclic
piperazine derivatives has been published in this regard (2). However these molecules have been only partially
characterized, hence the synthesis of selected 1,1-diphenyltetrahydro-1H-oxazolo[3,4-a]pyrazin-3(5H)-one
derivatives, bearing different substituents in 7-position, was undertaken aimed at defining the structural figures
relevant for NPSR antagonist activity.
The synthesized compounds were tested in vitro in a calcium mobilization assay, performed using HEK293 cells
expressing the recombinant mouse NPS receptor (HEK293mNPSR).
Tested compounds showed to inhibit NPSR with KB activity in the nanomolar range and, in particular, it has been
demonstrated that the CH2 linker in the side chain of the reference compound SHA 68 is not crucial for the
potency.
References:
(1) Xu, Y. L.; Reinscheid, R. K.; Huitron-Resendiz, S.; Clark, S. D.; Wang, Z.; Lin, S. H.; Brucher, F. A.; Zeng, J.; Ly, N. K.;
Henriksen, S. J.; de Lecea, L.; Civelli, O. Neuron. 2004, 43, 487-497.
(2) Yanan Z.; Brian P. G.; Hernán A. N.; Scott P. R. Bioorg. Med. Chem. Lett. 2008, 18, 4064-4067.
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123
OVERACTIVE BLADDER (OAB): A NEW THERAPEUTIC APPROACH.
1-PIPERAZINYLCARBOXAMIDES AND ANALOGUES AS
ALLOSTERIC ANTAGONISTS AT MGLU5 RECEPTOR
Carlo Riva, Carlo De Toma, Matteo Longhi, Ilaria Bettinelli, Katia Karamfilova, Davide Graziani, Gianni Motta,
Elena Poggesi, Amedeo Leonardi.
Drug Discovery – Recordati SpA
Overactive Bladder (OAB) is, in a societal perspective, a very onerous pathology, taking into account both direct
and indirect costs 1 . Thus, looking for new pharmacological treatments of OAB, as a replacement of antimuscarinic
drugs, which are not devoid of important side effects, could be an important issue.
MGlu5 receptors have been shown to play a role in the voiding reflex and bladder nociception. Experimental data
suggest that mGlu5 receptors play facilitator roles in the processing of afferent input from the urinary bladder, and
that central rather than peripheral mGlu5 receptors appear to be responsible 2 .
On these bases a new project was started, looking for mGluR5 allosteric antagonists (inverse agonist) by a
multidisciplinary approach, using as a starting point the structures of MTEP and MPEP (Figure 1).
.
Figure 1
A series of 1-piperazinylpropargylamides and closed analogues was synthesized with the aim of defining the main
features of the pharmacophore and the peculiar chemical groups, which could afford affinity for the mGlu5
receptor and selectivity vs. the mGlu1 receptor.
(1) Ganz, M.L.; Smalarz, A.M.; Krupski, T.L., Anger, J.T., Hu, J.C.; Wittrup-Jensen, K.U.; Pashos, C.L., Urology, 2009, Dec
24 in print – available on-line.
(2) Hu, Y.; Dong, L.; Sun, B.; Guillon, M.A.; Burbach, L.R.; Nunn, P.A.; Liu, X.; Vilenski, O.; Ford, A.P.D.W.; Zhong, Y.;
Rong, W., Neuroscience Letters, 2009, 450 (1), 12-17.
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124
NOVEL BIS(7)-TACRINE DERIVATIVES AS MULTITARGET
LIGANDS: FOCUS ON ANTI-CHOLINESTERASE AND ANTI-
AMYLOID ACTIVITIES
Maria Laura Bolognesi, Manuela Bartolini, Francesca Mancini, Gianpaolo Chiriano, Luisa Ceccarini, Michela
Rosini, Andrea Milelli, Vincenzo Tumiatti, Vincenza Andrisano, Carlo Melchiorre
Department of Pharmaceutical Science, University of Bologna , Via Belmeloro 6, 40126 Bologna, Italy
A vast array of drug discovery research focuses on the development of innovative drugs targeting Alzheimer's
disease (AD) and related dementias. However, despite several lead candidates have progressed to AD pre-clinical
testing in the last decade, to date none has been successful in late-stages clinical trials. Beta-secretase (BACE-1)
inhibition has been a strategy actively pursued, although progress in moving inhibitors to the clinic has been slow,
partly as a consequence of its aspartic proteinase character, which hampers the development of potent, selective
and brain-permeable compounds. At present, however, AD pharmacotherapy still relies on acetylcholinesterase
inhibitors donepezil, rivastigmine and galantamine. In this scenario, we and others envisaged the development of
Multi-Target Directed Ligands as an innovative and effective strategy to face the multifactorial etiopathogenesis of
AD (1). This strategy, embodied on single chemical entities able to simultaneously modulate multiple targets
involved in the neurodegenerative cascade, has proven particularly fruitful in the last couple of years and allowed
to discover several promising anti-Alzheimer's drug candidates.
Starting from this consideration, we have developed a new series of dimeric ligands derived from the lead
candidate bis(7)-tacrine, for which a multifunctional profile has been disclosed (2). The effect of replacing the
heptamethylene chain connecting the two monomeric tacrine units with polyaromatic spacers was evaluated at
three validated drug discovery targets, namely cholinesterases (acetyl- and butyrylcholinesterase) and amyloid
formation (BACE-1) and aggregation.
(1) Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini, M.; Melchiorre, C. J. Med. Chem. 2008,
51, 347-372.
(2) Li, W.; Mak, M.; Jiang, H.; Wang, Q.; Pang, Y.; Chen, K.; Han, Y. Neorotherapeutics 2009, 6, 187-201.
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DISCOVERY OF NEW POTENTIAL γ-SECRETASE MODULATORS
FOR THE TREATMENT OF ALZHEIMER’S DISEASE
Dario Ambrosini*, Gianfabio Giorgioni*, Piera Sozio, § Laura S.Cerasa, § Antonio Di Stefano §
Amelia Cataldi, ç Susi Zara. ç
*Scuola di Scienze del Farmaco e dei Prodotti della Salute, via S. Agostino 1, 62032, Camerino, Italy.
§ Dipartimento di Scienze del Farmaco, Università “G. D’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy.
ç Dipartimento di Biomorfologia, Università “G. D’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy.
125
The presence of Aβ-peptide containing plaques in the brain, is a hallmark of Alzheimer’s disease (AD). Several
studies have suggested a pathogenic role in AD for Aβ42, the longer form of the two Aβ-peptides, which has a
strongest tendency to deposit insoluble plaques in the AD brain. Aβ peptides are produced from β-Amyloid
Precursor Protein (APP) through the sequential cleavage by two proteases, namely β-secretase and γ-secretase.
Firstly, β-secretase produces a C-terminal fragment of APP (CTFβ), then γ-secretase cleaves the CTFβ to produce
Aβ peptides of different lengths. 1
Preventing the formation or intracellular deposition of Aβ42-peptide represents a strategy to stop or slow down the
progression of the AD. At this purpose, inhibition of γ-secretase has proven to be an interesting target for drug
discovery. 2 One of the first compounds which efficaciously inhibited Aβ production in animal models of AD
blocking γ-secretase activity was LY411575. 3 It has been demonstrated that inhibition of γ-secretase reduce Aβ in
a murine model of AD, but has potentially undesirable biological effects, most likely due to of the inhibition of
Notch cleavage, a transmembrane receptor involved in regulating cell-fate decision. 4 Thus, selective inhibition of
Aβ42 production seems to be a promising alternative to the complete inhibition of γ-secretase activity. The pursuit
of such a result was made possible by the surprising finding that certain non-steroidal anti-inflammatory drugs
(NSAIDs), including flubiprofen, display preferential Aβ42-lowering activity without affecting Notch cleavage. 5
The proposed mechanism for this activity is an allosteric modulation of γ-secretase activity, the enzyme
responsible for the formation of Aβ.
Starting from these data we proposed the synthesis of new LY411575 analogues that were designed to function as
potential γ-secretase modulators able to penetrate the blood-brain barrier, the synthesis and preliminary
pharmacological evaluation will be discussed.
(1) Olson, R.E. et al. Curr. Topics Med. Chem., 2008, 8, 17.
(2) a) Churcher I. et al. Curr. Pharm. Des., 2005, 11, 3363; b) Kornilova A.Y., et al. Annu. Rep. Med. Chem., 2003, 38, 41; (c)
John V. et al. J. Med. Chem., 2003, 46, 4625; (d) Josien H. Curr. Opin. Drug Discov. Devel., 2002, 5, 513.
(3) a)Wu, J.; et al. Int. Patent Appl. WO 9828268, 1998. (b) Audia, J. E.; et al. Int. Patent Appl. WO 2000019210, 2000.
(4) Wong G.T. et al. J. Biol. Chem., 279, 2004, 12876–12882.
(5) Czirr E. et al. Neurodegenerative Diseases, 2006, 3, 298.
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WITHDRAWN
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PUTATIVE GUANOSINE RECEPTOR IDENTIFICATION IN RAT
BRAIN BY THE INNOVATIVE EU EU-GTP GTP BINDING ASSAY
Michela Buccioni, , Diego Dal Ben, Catia Lambertucci, Carmen Lammi, Gabriella Marucci, Ajiroghene Thomas,
School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, 62032 Camerino, Italy Italy.
The purine nucleoside guanosine anosine possesses many trophic effects, including promotion, division, and growth of
astrocytes and other neuron-like like cells. This nucleoside seems to act as signalling molecule via the activation of
specific membrane receptors. 1 Competition studies, perfor performed med by Traversa et al. on rat brain membranes,
confirmed that [ 3 H]-guanosine guanosine site was distinct from the well characterized ATP and adenosine binding sites. 2
Starting from these observations and aimed at developing a new assay that allows to evaluate the po potency of
ligands at the putative guanosine receptor, Eu Eu-GTP GTP assay was performed in rat brain. This technique, using the
principle of the [ 35 S]GTPgammaS binding assay, replaces the radioactive material with Eu Eu-GTP and exploits the
unique fluorescence properties ties of Europium lanthanide chelate.
Furthermore, since potent and selective ligands are highly needed for the new putative guanosine receptor
characterization, new molecules were synthesized and tested using Eu Eu-GTP assay.
The results showed that some new compounds are able to activate the putative guanosine receptor with a potency
higher than that of guanosine itself. On the other hand, this functional assay allows to assume the presence in rat
brain of a GPCR activated by guanosine.
References:
(1) Caciagli, F.; Illes, P. Purinergic Signal. 2005, 1, 297-298.
(2) Traversa, U.; Bombi, G.; Camaioni Camaioni, E.; Macchiarulo, A.; Costantino, G.; Palmieri, C.; Caciagli Caciagli, F.; Pellicciari, R. Bioorg.
Med. Chem. 2003, 11(24), 5417-5425. 5425.
RosariaVolpini, Gloria Cristalli
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128
PARALLEL SYNTHESIS AND EVALUATION OF 2,5-DIAMINO-1,4-
BENZOQUINONES AS A NOVEL CLASS OF BIFUNCTIONAL ANTI-
PRION COMPOUNDS
Salvatore Bongarzone, 1, 2 Hoang Ngoc Ai Tran, 3 Andrea Cavalli, 4,5 Marinella Roberti, 4 Paolo Carloni, 1,2,6,7
Giuseppe Legname, 2,3 and Maria Laura Bolognesi 4
1 Statistical and Biological Physics Sector, International School for Advanced Studies (SISSA), 34151 Trieste, Italy.
2 SISSA-Unit, Italian Institute of Technology, 34151 Trieste, Italy.
3 Neurobiology Sector, International School for Advanced Studies (SISSA), 34151 Trieste, Italy.
4 Department of Pharmaceutical Sciences, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
5 Department of Drug Discovery and Development, Italian Institute of Technology, 16163 Genova, Italy.
6 CNR-INFM-DEMOCRITOS Modeling Center for Research in Atomistic Simulation, 34151 Trieste, Italy.
7 German Research School for Simulation Sciences GmbH, Forschungzentrum Jülich GmbH - RWTH Aachen University.
Prion diseases, also known as transmissible spongiform encephalopathies (TSE), are fatal neurodegenerative
disorders of the central nervous system. The main molecular mechanism underlining TSE is based on the aberrant
misfolding of the cellular form of the prion protein (PrP C ) into its pathological counterpart denominated PrP Sc . To
date there are no identified therapies. One therapeutic strategy against this disease is focused on the stabilization of
the PrP C in order to prevent its conversion to PrP Sc (1). PrP Sc is involved in fibrillation processes and thus play a
pivotal role in the pathogenesis of TSEs. Crucial elements mediating fibrillation event are protein-protein
interactions (PPIs). Although medicinal chemistry approaches considering PPIs as drug target are daunting
challenge, recent studies showed that small molecules bearing homodimeric structure properly spaced have shown
possible therapeutic effect (2). Building on the bivalent approach, we have recently reported that 2,5-diamino-1,4benzoquinone
(BQ) linked with two phenyl rings by a spacer displayed remarkable anti-prion activity in a cellular
model (EC50 = 0.87 µM) (3). This allowed us to propose that the planar BQ scaffold might be considered as a
privileged motif in modulating PPIs and as a promising central core in the search for bifunctional anti-prion
compounds. Starting from these results, a small library featured a BQ nucleus as central core, with two spacers in
position 2 and 5 connected to two terminal moieties was designed. Designed entries were made by parallel
synthesis, and evaluated against prion infection. Some of them have shown inhibition against PrP Sc aggregation in
ScGT1 cell line. The most promising anti-prion compounds (quinoline derivatives) have shown low toxicity and
capability to inhibit prion fibril formation in vitro.
PrP C PrP Sc fibril of PrP Sc
O O
H
Cl N
O
H H
O
N Cl
H
O O
EC50= 0.87 µM
(1) Caughey, B.; Baron, G. S. Nature 2006, 443, 803-810
(2) May, B. C. H.; Fafarman, A. T.; Hong, S. B.; Rogers, M.; Deady, L. W.; Prusiner, S. B.; Cohen, F.; .Proc. Natl. Acad. Sci. U. S. A. 2003,
100, 3416-3421.
P-16
N
Cl
H
N
N
H
O
O
H
N
N
H
N
Cl

AMINOPYRIMIDINES AS INHIBITORS OF THE WNT SIGNALLING
PATHWAY
Fabio Del Bello, a Aniket Farande, a Mario Giannella, a Alessandro Piergentili, a Wilma Quaglia, a Arianna Nencini, b
Russell Thomas, b Maurizio Varrone, b Tiziana Benicchi, b Giovanni Benedetti, b Alessandra De Robertis, b
Silvia Valensin b
129
a Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, via S. Agostino 1, 62032 Camerino, Italy
b Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy
The aberrant activation of the Wnt signalling pathway is believed to be a major driving force in a broad range of
pathological conditions and is common in many types of human tumors. Inhibition of the pathway has been shown
to block tumor growth giving great therapeutic potential to drugs targeting this pathway. 1
In a program aimed at the identification of small molecules inhibitors of the Wnt pathway for the treatment of
glioblastoma (GBM) we identify a series of aminopyridines which specifically inhibit the pathway without
affecting cell viability. This series of compounds has been expanded with the synthesis of a focused set of
aminopyrimidines with two points of diversity. All compounds have been tested for their activity on the Wnt
pathway using DBTRG cells stably transfected with TCF-Luciferase and TA-Renilla. The TCF-Luc reporter gene
specifically responds to Wnt stimuli while the Renilla readout is Wnt independent and can be used as cell viability
readout. In addition, in order to identify potential ADME liabilities, all compounds were tested for their in vitro
solubility, metabolic stability and permeability.
(1) Barker, N.; Clevers, H. Nat. Rev. Drug Discov. 2006, 5, 997-1014.
This work was supported by the Monte dei Paschi di Siena Foundation Award
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130
INSECTICIDAL HETEROLIGNANS – IN VIVO SAR STUDY OF
POTENT TUBULINE POLYMERIZATION INHIBITORS WITH
ACTIVITY AGAINST CHEWING PESTS
Jens Frackenpohl a , Isabelle Adelt b , Horst Antonicek b , Christian Arnold b , Nicole Blaha a , Sabine
Hohmann a , Elisabeth Peschel a , Thomas Schenke b , Robert Velten b , Hans-Christoph Weiss c
a Bayer CropScience AG, Industriepark Höchst, G836, D-65926 Frankfurt a. Main
b Bayer CropScience AG, Alfred-Nobel-Strasse 50, D-40789 Monheim
c Currenta GmbH, Chemiepark Leverkusen, Q18, D-51368 Leverkusen
Starting from natural product podophyllotoxin substituted heterolignans were identified with promising
insecticidal in vivo activity. The impact of substitution in each segment of the core structure was
investigated in a detailed in vivo-SAR study, and variation of substituents in both aromatic moieties
afforded promising derivatives with broad insecticidal activity against lepidopteran and coleopteran
species. In vitro measurements supported by modeling studies indicate that heterolignans investigated in
our study act as potent tubuline polymerization inhibitors interacting with the colchicine-binding site.
Various unprecedented substituents have been introduced to fully explore the structure-insecticidal
activity relationship. Thus, insect specific structure-activity effects were observed showing that the
insecticidal SAR described herein differs from reported cytotoxicity studies.
O
O
A
O
OH
H
B C O
D
O
H
O
O
Podophyllotoxin Deoxypodophyllotoxin
[1] J. Frackenpohl et al., WO 05097802, 2005.
[2] J. Frackenpohl et al., Bioorg. Med. Chem., 2009, 17, 4160-4184.
O
O
O
O
H
H
O
O
O
O
O
P-18
6
5
7 8
O
H
N 4
O
9
3
O
1
O
O
O
O
O
H
N
O
O
O

TARGETING PROTEIN PROTEIN-PROTEIN PROTEIN INTERACTIONS FOR NOVEL
ANTICANCER THERAPIES
THERAPIES: : MOLECULAR DYNAMICS OF SMALL
MOLECULES INHIBITING THE p53-MDM2 MDM2 COMPLEX.
Nicola Giacchè Giacchè, Antonio Macchiarulo, Roberto Pellicciari.
Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia, via del Liceo 1, 06123 Perugia.
MDM2 is an oncogenic protein that regulates the activity and stability of p53. The evidence that more than 50% of
human cancers show altered regulations of p53 and overexpressed levels of MDM2, has fu fuelled the search for new
therapeutic agents that could rescue p53 from the inhibition of MDM2. 1-2 Accordingly, during the last decade,
diverse small molecules have been disclosed as able to disrupt the interaction between MDM2 and p53. On this
basis, experimental imental and computational approaches have been developed to study the interaction of small molecules
with the p53 binding cleft of MDM2, aimed at facilitating the bench to bedside translation of new potent and
selective chemical entities as novel anticance anticancer therapies. 3
In this framework, as continuation of our efforts in the field, 4-5 we report the results of molecular dynamic
simulations carried out to study the conformational transitions of MDM2 in response to small molecule binding.
Our study points out a conditional active role of the NN-terminal
region (1-25 25 residues) of MDM2 in the molecular
recognition of inhibitors, that is dependent on the specific chemical class of the ligand. The results obtained will
prove useful to drive medicinal chemistry in de designing signing new MDM2 inhibitors with improved potency and
selectivity.
(1) Vogelstein, B.; Lane, D.; Levine, AJ. Nature 2000, 408, 307-310; 310; (2) Toledo, F.; Whal, GM. Nat. Rev. Cancer 2006, 6,
909-923; (3) Patel, S.; Player, MR. Expert Opin. Investig. Drugs 2008, 17, 1865-1882; 1882; (4) Macchiarulo, A.; Giacchè, N.;
Carotti, A.; Baroni, M.; Cruciani, G.; Pellicciari, R. J. Chem. Inf. Model. 2008, 48, 1999-2009; 2009; (5) Carotti, A.; Macchiarulo, A.;
Giacchè, N.; Pellicciari, R. Proteins 2009 2009, 77, 524-535.
Acknowledgements:
This work was supported by the European Union. FP6 PRIORITY LSH LSH-2005-2.2.0-8: Small-ligand ligand libraries: improved tools
for exploration and prospective anti-tumour tumour therapy. DePPICT Project (Designing Therapeutic Protein Protein-Protein Inhibitors for
Brain n Cancer Treatments) Contract number: LSHC LSHC-CT-2007- 037834 (http://www.deppict.eu/home.jsp)
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132
WITHDRAWN
P-20

DESIGN AND SYNTHESIS OF Smo ANTAGONISTS FOR TREATMENT
OF CANCER
133
Annette Bakker, Marta Bellini, Giovanni Benedetti, Chiara Caramelli, Pietro Ferruzzi, Simone Galeazzi, Giacomo
Minetto, Gal.la Pericot Mohr, Andrea Nuzzi, Alessandro Panico, Francesco Russo and Russell Thomas
Medicinal Chemistry Department, Siena Biotech S.p.A., Strada del Petriccio e Belriguardo, 35. 53100, Siena, Italy
gminetto@sienabiotech.it.
The Hedgehog pathway plays an important role in the embryonic patterning and development of many tissues and
somatic structures as well as maintaining and repairing mature tissues in adults. Uncontrolled activation of this
pathway has been implicated in several cancers including glioblastoma, melanoma, basal cell carcinoma and
breast, lung, prostate and pancreatic cancers. The aberrant activation of Hedgehog signaling in several cancers has
made it an attractive target for anticancer drug discovery (1-2).
Most of the medicinal chemistry efforts have been directed towards targeting Smo, a transmembrane protein that
plays a central role in modulating Gli transcription factors and initiating transcription of Hh target genes. A
number of Smo inhibitors have been reported and they can be classified as cyclopamine analogues or synthetic
small molecule antagonists (3).
Different classes of molecules have been designed in Siena Biotech as Smo inhibitors for treatment of
glioblastoma. Great attention has been put towards the physicochemical properties of such molecules in order to
facilitate their subsequent optimization.
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134
DESIGN, SYNTHESIS, AND BIOLOGICAL EVALUATION OF
NAPHTHALENE DIIMIDES DERIVATIVES AS ANTICANCER AGENTS
A. Milelli 1 , A. Minarini 1 , C. Melchiorre 1 , M. Micco 1 , E. Simoni 1 , G. Zuccari 1 , L. Raffaghello 3 , C. Stefanelli 2 ,
M. Zini 2 , V. Tumiatti 1
1 Department of Pharmaceutical Sciences, Via Belmeloro 6, 2 Department of Biochemistry
“G. Moruzzi”, Via Irnerio 48, Alma Mater Studiorum, University of Bologna, 40126 Bologna, Italy
3 Laboratory of Oncology, Giannina Gaslini Institute, Genova, Italy.
The search for novel chemotherapeutic agents and approaches to cancer treatment is an active research field
stimulated by the discovery of new biological targets and by the possibility of obtaining new drugs without serious
and undesirable side effects. Several anticancer agents have been developed so far based on different mechanism
of action; despite this, intercalator agents still remain an interesting class of molecules to focus on. In particular, in
the literature there are several examples of Naphthalimmide and 1,4,5,8-Naphtalentetracaboxylic diimide
derivatives as intercalating and anticancer agents. It is well-known that, at physiological pH, protonated
polyamines interact strongly with the phosphate residues of DNA. Therefore, the inclusion of these basic
functionalities, on an intercalating moiety, may improve the interaction with DNA structure.
Recently, we reported on the design and synthesis of substituted Naphtalen(di)immides as anticancer agents (1).
Among the several developed compounds, 1 and 2 were the most interesting derivatives showing the ability to
bind DNA, trigger caspase activation, cause accumulation of p53 protein, down-regulate the survival kinase AKT,
cause a decrease of ERK1/2 and, inhibit ERK’s phosphorylation.
OCH 3
H
N
O
N
n
O
1: n = 1
2: n = 2
The aim of the present work is to further investigate the promising biological profile of 1 and 2, and the role of the
2-methoxy substituents on the benzyl moieties. For this purpose several analogues, with different substituents on
the two aromatic rings, were synthesized and evaluated for their antiproliferative activity.
This research was supported by grants from MUR, the University of Bologna, and Polo Scientifico-Didattico di Rimini. We
thank the National Cancer Institute for the anticancer assays.
(1) Tumiatti, V., Milelli, A., Minarini, A., Micco, M., Gasperi Campani, A., Roncuzzi, L., Baiocchi, D., Marinello, J.,
Capranico, G., Zini, M., Stefanelli, C., Melchiorre C. J. Med. Chem. 2009, 52, 7873-7.
O
N
O
P-22
n
H
N
OCH 3

SULFONATE BILE ACID DERIVATIVES AS POTENT AND
SELECTIVE MODULATORS OF TGR5, A NEW TARGET FOR
DIABESITY
Paola Sabbatini, 1 Antimo Gioiello, 1 Antonio Macchiarulo, 1 Emiliano Rosatelli, 1 Gianni Rizzo, 2 Luciano Adorini, 2
Johan Auwerx, 3 Charles Thomas, 3 Roberto Pellicciari. 1
1) Università degli Studi di Perugia, Dipartimento di Chimica e Tecnologia del Farmaco, Via del Liceo, 1, 06100 Perugia,
Italy. 2) Intercept Italia, Via P. Togliatti, 22,06073 Corciano, Italy. 3) Institut Clinique de la Souris, 67404 Illkirch, France.
135
TGR5 is a bile acid (BA) membrane receptor, G-protein coupled to the adenylate cyclase. On the basis of
sequence similarity studies, it belongs to the group of MECA (melanocortin, endothelial, cannabinoid and
adenosine) receptors, inside the rhodopsine-like family of GPCRs. Recent years have witnessed a growing interest
in TGR5 as an attractive target for obesity and type-2-diabetes, due to its roles in the regulation of glucose
metabolism and energy homeostasis. 1 These effects are linked to the ability of the receptor to stimulate type 2
iodothyronine deiodinase (D2) activity in brown adipose tissue (BAT) and muscle, as well as induce the release of
glucagon-like peptide 1 (GLP-1) in enteroendocrine cells. 1
In this scenario, we have been committed toward the identification of novel BA derivatives as potent and selective
agonists for TGR5. Our efforts have hitherto resulted in the disclosure of S-EMCA (INT-777), a compound that
has been instrumental to provide the first proof-of-concept that the pharmacological modulation of TGR5 may
indeed represent a novel viable strategy for the treatment of type 2 diabetes. 2,3 On the basis of our preliminary
QSAR study, 4 and as a continuation of our work in the field, we have further investigated the effect of
modifications on the BA side chain. In particular, in this communication we report the design, synthesis and
preliminary biological appraisals of a new series of BA derivatives, bearing bioisosteric substitution of the
carboxylic group with sulfonate moieties.
References:
(1) Thomas, C.; Pellicciari, R.; Pruzanski, M.; Auwerx, J. Schoonjans, K. Nat. Rev. Drug Disc. 2008, 7(8), 678-693.
(2) Pellicciari, R.; Gioiello, A.; Macchiarulo, A.; Thomas, C.; Rosatelli, E.; Natalini, B.; Sardella, R.; Pruzanski, M.; Roda,
A.; Pastorini, E.; Schoonjans, K.; Auwerx, J. J. Med. Chem. 2009, 52 (24), 7958–7961.
(3) Thomas, C.; Gioiello, A.; Noriega, L.; Strehle1, A.; Oury1, J.; Rizzo, G.; Macchiarulo, A.; Yamamoto, H.; Mataki, C.;
Pruzanski, M.; Pellicciari, R.; Auwerx, J.; Schoonjans, K. Cell Met. 2009, 10(3), 162-164.
(4) Macchiarulo, A.; Gioiello, A.; Thomas, C.; Massarotti, A.; Nuti, R.; Rosatelli, E.; Sabbatini, P.; Schoonjans, K.;
Auwerx, J.; Pellicciari, R. J. Chem. Inf. Model. 2008, 48 (9), 1792–1801.
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136
DIKETOPIPERAZINE-BASED LIGANDS OF PRION PROTEIN
Maria Laura Bolognesi, b Matteo Staderini, a Hoang Ngoc Ai Tran, d Alessandra Monaco, a Salvatore Bongarzone, ce
Xevi Biarnés, ce Pilar López-Alvarado, a Nieves Cabezas, a Paolo Carloni, ce J. Carlos Menéndez, a and Giuseppe
Legname de
aDepartamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad Complutense, 28040 Madrid,
Spain. bDepartment of Pharmaceutical Sciences, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126
Bologna, Italy. cStatistical and Biological Physics Sector, dNeurobiology Sector, International School for Advanced Studies
(SISSA), eSISSA-Unit, Italian Institute of Technology, 34014 Trieste, Italy.
Prion diseases, or transmissible spongiform encephalopathies (TSEs) are a family of invariably fatal
neurodegenerative disorders for which no effective curative therapy currently exists. A common feature of TSEs is
the deposition of insoluble aggregates of disease-associated prion protein (PrP Sc ), the post-translationally refolded
and partially protease-resistant isoform of normal cellular prion protein (PrP C ).(1)
The interactions between proteins are important for many biological functions and prion diseases have been linked
to protein folding problems which lead to the build-up of insoluble protein plaques in the brain or other organs.
The ability to design novel ligands that inhibit such interactions remains one of the major challenges in
contemporary biomedical science.(2)
We aimed to identify anti-prion lead compounds with unprecedented molecular frameworks. As the result of the
structural similarity of diketopiperazines (DPKs) to peptides, their appearance in biologically active natural
products has inspired the use of DKPs to circumvent the limitations of peptides. We propose DKPs as modulators
of critical protein-protein interactions in prion disease, inhibiting the conversion of PrP C to PrP Sc .
For the design of our compounds, we noticed that most anti-prion molecules possess a symmetrical bifunctional
structure, consisting of two moieties joined via an appropriate spacer. Since we consider the DKP nucleus as a
privileged structure in the modulation of protein-protein interactions, we have synthesized some dimeric
compounds by the three-component double condensation of 1,4-diacetyl-2,5-piperazinedione with aromatic
aldehydes. To study the importance, if any, of an appropriate planar conformation some monoreduced and a
saturated derivative were also synthesized.
A cell screening assay was used to test anti-prion activity across the synthesized compounds. The ability to reduce
PrP Sc concentrations in scrapie-infected mouse hypothalamus (ScGT1) cells was determined from Western blot
densitometry of the PK-resistant PrP Sc . We report the identification of a novel bifunctional DKP derivative, which
exhibited activity in the low micromolar range against prion replication in ScGT1 cells, while showing low
toxicity.
References
(1) Hu, W.; Kieseier, B.; Frohman, E.; Eagar, T. N.; Rosenberg, R. N.;Hartung, H.-P.; St€uve, O. Prion proteins: physiological
functions and role in neurological disorders. J. Neurol. Sci. 2008, 264, 1–8.
(2) Breinbauer, R.;, Vetter, I. R;. Waldmann, H.. From protein domains to drug candidates-natural products as guiding
principles in the design and synthesis of compound libraries. Angew. Chem. Int. Ed. 2002, 41, 2878 – 2890.
P-24

SOUNDS OF SILENCE: INNOVATIVE APPROACH FOR THE
IDENTIFICATION OF NOVEL GPCR-MODULATOR CHEMICAL
ENTITIES
Stephan Schann, Stanislas Mayer, Mélanie Frauli, Christel Franchet, Pascal Neuville
Domain Therapeutics, BioParc, Boulevard Sebastien Brant, F-67400 Illkirch, France
137
Allosteric modulators (AMs) represent an emerging and promising approach for the regulation of G-protein
coupled receptors (GPCRs), the best family of drug targets with nearly half of the current market for therapeutic
agents. AM’s use is associated with major advantages such as subtype selectivity, saturable effects, mimicking of
physiological response and more tractable chemistry. To date, two GPCR AMs have entered the market:
Cinacalcet, a positive allosteric modulator (PAM) of calcium sensing receptor for the treatment of
hyperparathyroidism and Maraviroc, a negative allosteric modulator (NAM) of CCR5 for the treatment of HIV. 1,2
This relatively low number can be explained by the lack of technology specifically dedicated for their discovery.
The standard process used in the Pharmaceutical industry consists in performing an HTS screening with a
functional assay using specific conditions and a single readout. This process undersamples the chemical space
surrounding potential therapeutic candidate for a chosen GPCR. 3 At Domain Therapeutics we are using a different
strategy consisting in identifying first a broad set of GPCR binders using a FRET-based binding assay 4 and then
characterizing these binders with multiple functional test paradigm to make sure no active AM is missed. This
process enables detection of PAMs, NAMs but also silent allosteric modulators (SAMs), which are binders devoid
of any functional activity. These SAMs constitute a class of molecules not detectable with functional assay and
that represent a unique source of chemical diversity.
References:
(1) Conn, PJ.; Christopoulos, A.; Lindsley, CW. Nat. Rev. Drug Disco. 2009, 8, 41-54.
(2) Bridges, TM.; Lindsley, CW. ACS Chem. Biol. 2008, 3(9), 530-41.
(3) Leach, K.; Sexton, PM.; Christopoulos, A. TiPS 2007, 28(8), 382-9.
(4) Tahtaoui, C.; Guillier, F.; Klotz, P.; Galzi, JL. ; Hibert, M.; Ilien, B. J. Med. Chem. 2005, 48, 7847-59.
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LIST of PARTICIPANTS

Allegretti Marcello Italy 43
Ambrosini Dario Italy 125
Angeli Piero Italy
Baringhaus Karl-Heinz Germany 63
Bentley Jonathan UK 109
Berk Barkin Turkey 101, 113
Bolognesi Maria Laura Italy 25, 108, 124, 128, 136
Bongarzone Salvatore Italy 128, 136
Booth Raymond G USA 102
Borza István Hungary
Boss Christoph Switzerland 85
Bouvier Michel Canada 39
Bovino Clara Italy
Brandt Wolfgang Germany
Buccioni Michela Italy 115, 116, 127
Cano Montserrat Spain
Cappellacci Loredana Italy 114
Carrieri Antonio Italy 104, 118
Congreve Miles S UK 61
Costanzi Stefano USA 101, 113
Cristalli Gloria Italy 115, 116, 117, 122, 127
Dal Ben Diego Italy 115, 116, 117, 122, 127
De Amici Marco Italy 69
Del Bello Fabio Italy 104, 119, 120, 129
Di Fabio Romano Italy 79, 81
Donati Daniele Italy 22
Dukat Malgorzata USA 106
Enzensperger Christoph Germany 51
Farande Aniket Italy 119, 120, 129
Farande Yogita Italy 120
Farouz Francine USA 87
Forlani Roberto Italy
139

140
Frackenpohl Jens Germany 130
Froestl Wolfgang F Switzerland
Gaviraghi Giovanni Italy 93
Ghelfi Francesca Italy 118, 119
Ghiron Chiara Italy 35
Giacchè Nicola Italy 131
Giacobbe Dania Italy 115, 122
Giannella Mario Italy 104, 119, 120, 129
Giardina Giuseppe Italy
Giardinà Dario Italy
Giorgioni Gianfabio Italy 125
Gitto Rosaria Italy 121
Glennon Richard A USA 24
Goller Andreas H Germany
Gualtieri Fulvio Italy
Hacksell Uli USA 17
Hubbard Roderick E UK 31
Ijjaali Ismail France 100
Kerns Edward H USA 33
Lankau Hans J Germany
Lange Udo E W Germany
Leeson Paul D UK 37
Leurs Rob The Netherlands 21, 45, 103
Li Xue-Juan P.R. China 107
Makriyannis Alexandros USA 27, 53
Marchioro Carla Italy
Martinelli Adriano Italy 23
Mason Jonathan S Denmark 71
Melchiorre Carlo Italy 25, 108, 124, 134
Merlo Pich Emilio Italy 79
Milelli Andrea Italy 124, 134

Minetto Giacomo Italy 133
Mogemark Mickael Sweden
Mohr Klaus Germany 69
Morphy Richard UK 73
Mosti Luisa Italy
Moureau Florence Belgium
Neumeyer John L USA
Nilsson Karolina Sweden
Ofner Silvio Switzerland
Panmand Deepak S Italy 115, 117
Pegoraro Stefano Germany
Pellicciari Roberto Italy 49, 131, 135
Piergentili Alessandro Italy 104, 119, 120, 129
Pigini Maria Italy 104, 118, 119, 120
Piomelli Daniele USA 55
Porter Roderick Italy 81
Portoghese Philip S USA 59
Quaglia Wilma Italy 104, 119, 120, 129
Reid Mark UK
Riva Carlo Italy 123
Roecker Anthony J USA 83
Ronzoni Silvano Italy
Rosini Michela Italy 108, 124
Sabbatini Paola Italy 135
Saunders John USA
Schann Stephan France 47, 137
Schaus John M USA
Schell Peter Sweden
Schoen Uwe Germany
Schulte Marvin K USA 67
Selent Jana Spain 110
Simoni Elena Italy 108, 134
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142
Smit Martine J The Netherlands 45
Smits Rogier A The Netherlands 103
Staderini Matteo Spain 136
Svenningsson Per J L Sweden 65
Szentirmay Eva Hungary
Thomas Ajiroghene Italy 116, 127
Thomas Russell J Italy 20, 91, 118, 119, 129, 133
Timmerman Henk The Netherlands 26
Triggle David J USA 95
Tuccinardi Tiziano Italy 105
Tunici Patrizia Italy 89
Van der Schyf C. (Neels) J USA 75
Weber Lutz Germany
Youdim Moussa B H Israel 77
Zaman Guido J R The Netherlands 41

ACKNOWLEDGEMENTS
info@coneroazzurro.it
www.coneroazzurro.it
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Farmacia Angelini
Dr. Daniele Maria
Miips Informati tiica
Comune di Camerino
Comune di Matelica
Associazione
Corsa alla Spada
ARTELITO TELITO
SOLUZIONI LITOGRAFICHE RAFICHE
University of Camerino
Photos by Maurizio Brandi and Pierluigi Lana
Comune di Castelraimondo
Comunità Montana
di San Severino Marche
illuminazione
Fondaziion one
MA..SO..GI.. ..BA..