Peptide-Based Drug Design

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Targeted Therapeutic and Imaging Agents 277 15-aa-long N-terminal fragment of human ribonuclease I, in which arginine in position 4 is substituted for cysteine. In our experience, Cys-tag is compatible with polypeptides varying in length from 53 to 358 aa (Table 1), and can be used for site-specific conjugation of very different payloads (Table 2) without affecting protein or peptide functionality. We therefore expect that Cys-tag would provide a standardized approach to coupling therapeutic and contrast agents to various bioactive polyamides and will be translatable into the development of clinical products. We will briefly review accumulated experience in making and using Cys-tagged peptides and proteins, focusing on Cys-tagged human vascular endothelial growth factor (VEGF). We will discuss technical problems that might arise in using our platform for development of new conjugates for targeted delivery of therapeutic and diagnostic agents. 1.2. Cys-Tagged Proteins Plasmids for bacterial expression of proteins with N- or C-terminal Cystag have been described previously (1). They were made by inserting the coding sequence for Cys-tag (Lys-Glu-Ser-Cys-Ala-Lys-Lys-Phe-Gln-Arg-Gln- His-Met-Asp-Ser) and a Ser-Gly linker in the appropriate sites in the commercially available pET29 plasmid for bacterial expression of proteins under control of T7 promoter (Novagen, WI). So far, expression of Cys-tagged proteins in insect, yeast, or mammalian hosts has not been tested. Table 1 Peptide and Proteins Expressed with Cys-tag Total number of Number of Proteins and peptide amino acids native cysteines Ref. VEGFa 110–222 16–18 (1–7,15) EGFb 53 6 (6) EGF-SLTc 358 8 Unpublished Soluble FLT3 ligand (FLex) 154 6 Unpublished Annexin V 330 1 Unpublished Catalytically inactive fragment of anthrax lethal factor (LFn) 254 0 (5,7) a Several human VEGF121-based Cys-tagged proteins were constructed and tested, including VEGF121 dimer, truncated VEGF110 dimer, and single-chain (sc) VEGF comprising two 3-110 fragments of VEGF121 fused head-to-tail. b Cys-tagwasfusedtohumanEGFvia(G4S)3 linker. c EGF-SLT comprises Cys-tagged EGF fused to catalytic subunit of E. coli Shiga-like toxin.
278 Backer et al. Table 2 Payloads Conjugated to Cys-Tagged Proteins Payloads Applications Ref. Lipid/Liposome Drug delivery (3,5,7) Dendrimers Drug delivery (1) DOTA Targeted PET imaging (7) PEG-DOTA Targeted PET imaging (7,15) Targeted SPECT imaging Unpublished Targeted radiotherapeutics Unpublished HYNIC Targeted SPECT imaging (2,7) “Self-chelation” of 99mTc Targeted SPECT imaging (15) PEG Changing protein’s pharmacokinetics and immunogenicity (7) Fluorescent dyes Targeted imaging in whole animals (1,5,7) Tagging cells with active and accessible receptors in vivo (7) Receptor-mediated endocytosis in vitro (1,6,7) Biotin Targeted imaging with quantum dots Unpublished and ultrasound microbubbles Adapter protein Flexibility in constructing conjugates (5) Fibronectin Tissue culture scaffolds (6) To date, all Cys-tagged proteins and peptides were expressed in Escherichia coli BL21(DE3) strain. As usual, growth temperature, bacterial density for protein induction, concentration of inducer (IPTG), and duration of induction should be optimized for every protein. Almost all our Cys-tagged proteins were found in inclusion bodies. In our experience, recovery of proteins from inclusion bodies might be greatly improved by a combination of sequential washings and mild sonication prior to solubilization of inclusion bodies. Expression of proteins with Cys-tag adds an additional cysteine to the set of native cysteines, and therefore it might complicate the formation of native disulfide bonds. In general, protein refolding from a denatured state is achieved by slow removal of denaturing components, while maintaining Red- Ox environment using a mixture of reduced and oxidized forms of glutathione. While peptide is denatured, each cysteine can form a reversible disulfide bond either with other cysteine or with glutathione. Over time, as denaturing components are slowly removed, this process selects stable cysteine–cysteine disulfide bonds that correspond to thermodynamically favored properly folded peptide conformations. Importantly, since cysteine in Cys-tag does not have a “natural” partner, successful refolding leaves the C4 thiol group in a mixed disulfide with glutathione. The key point in refolding is to maintain a reasonable pace of
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Peptide-Based Drug Design
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METHODS IN MOLECULAR BIOLOGY TM Pep
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Preface Natural products chemistry
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Contents Preface...................
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Contributors Nikolinka Antcheva •
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Contributors xi Alessandro Tossi
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2 Otvos advance of computer power a
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4 Otvos see derivatives active in b
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6 Otvos was designed based on ligan
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8 Otvos 27. Borghouts, C., Kunz, C.
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10 Bulet Key Words: Invertebrate im
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12 Bulet 6. 5 �L or higher volume
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14 Bulet 2.5.2.1. MALDI-TOF-MS 1. M
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16 Bulet 7. Small-volume low-protei
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18 Bulet 3. Centrifuge between 8000
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20 Bulet internal diameter). Increa
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22 Bulet interest. As no instrument
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24 Bulet 3. Incubate the plates in
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26 Bulet bioactive peptides from th
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28 Bulet 21. Chernysh, S., Kim, S.I
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3 Sequence Analysis of Antimicrobia
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Sequence Analysis of Antimicrobial
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4 The Spot Technique: Synthesis and
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The Spot Technique 49 3. For amine
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The Spot Technique 51 2. Biotinylat
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The Spot Technique 53 the solutions
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The Spot Technique 55 solution. Ens
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The Spot Technique 57 (see Fig. 3)
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The Spot Technique 59 Fig. 5. Princ
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The Spot Technique 61 library, the
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The Spot Technique 63 7. Regenerati
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The Spot Technique 65 following rul
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The Spot Technique 67 20. Atherton,
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The Spot Technique 69 50. Bolger, G
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5 Analysis of A� Interactions Usi
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Analysis of Aβ Interactions 73 are
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Analysis of Aβ Interactions 75 Ana
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Analysis of Aβ Interactions 77 3.
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6 NMR in Peptide Drug Development J
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NMR of Peptides 89 usually require
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NMR of Peptides 91 limiting the siz
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NMR of Peptides 93 and STD NMR expe
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NMR of Peptides 95 The basis of the
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NMR of Peptides 97 Fig. 5. Overlay
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NMR of Peptides 99 Fig. 7. Schemati
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NMR of Peptides 101 4.4.2. Saturati
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NMR of Peptides 103 4.6. Diffusion
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NMR of Peptides 105 Fig. 13. Propos
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NMR of Peptides 107 protein prevent
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NMR of Peptides 109 6. Wuthrich, K.
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NMR of Peptides 111 45. Morris, K.
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NMR of Peptides 113 78. Aumailley,
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116 Copps et al. Fig. 1. Potential
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118 Copps et al. Fig. 2. Flowchart
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120 Copps et al. 10. In accordance
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122 Copps et al. Fig. 3. DSSP for g
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124 Copps et al. ingly with the sam
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126 Copps et al. 19. Essman, U., Pe
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128 Hilpert et al. Key Words: Scree
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130 Hilpert et al. Table 1 (Continu
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132 Hilpert et al. different natura
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134 Hilpert et al. wt A C D E F …
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136 Hilpert et al. methods primaril
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144 Hilpert et al. Table 3 Summary
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148 Hilpert et al. of substituting
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150 Hilpert et al. in models that c
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152 Hilpert et al. than control. Gi
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154 Hilpert et al. Table 4 Accuracy
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156 Hilpert et al. 25. Pini, A., Gi
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158 Hilpert et al. 55. Giacometti,
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9 Investigating the Mode of Action
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Proline-Rich Antimicrobial Peptides
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10 Serum Stability of Peptides Håv
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Serum Stability of Peptides 179 2.
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Serum Stability of Peptides 183 �
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11 Preparation of Glycosylated Amin
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Glycosylated Amino Acid Synthesis 1
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12 Synthesis of O-Phosphopeptides o
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Solid-Phase Synthesis of O-Phosphop
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13 Peptidomimetics: Fmoc Solid-Phas
Page 234 and 235: Peptidomimetics 225 O O R S N H Pep
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Page 240 and 241: Peptidomimetics 231 of peptidosulfo
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Page 258 and 259: Lipopeptides 249 2. Materials 2.1.
Page 260 and 261: Lipopeptides 251 Fig. 1. Structural
Page 262 and 263: Lipopeptides 253 8. To enable lipid
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Page 266 and 267: Lipopeptides 257 Fig. 2. Immunogeni
Page 268 and 269: Lipopeptides 259 8. A large plastic
Page 270 and 271: Lipopeptides 261 26. Nardin, E.H.,
Page 272 and 273: 264 Otvos response, synthetic pepti
Page 274 and 275: 266 Otvos Fig. 3. Schematic present
Page 276 and 277: 268 Otvos 3. Methods The main goal
Page 278 and 279: 270 Otvos 3.2.3. Purification and Q
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Page 302 and 303: Index A A� peptides aggregation a
Page 304 and 305: Index 297 phosphopeptides influenci
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Page 308 and 309: Index 301 peptidosulfonamide, disad
Page 310: Index 303 solid phase, 180, 214 sul
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Peptide-Based Drug Design

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