Peptide-Based Drug Design

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Analysis of Aβ Interactions 75 Analysis of A�–lipid interactions involves performing ultracentrifugation or size filtration chromatography of vesicles exposed to A�, CD spectroscopy, or surface plasmon resonance (SPR) technology. Chromatography and ultracentrifugation require several hours, during which time the peptides can further aggregate, complicating the interpretation of the data. CD indicates structural changes to the peptide upon interactions with lipids, but quantitative interpretation is difficult. SPR allows lipid-binding affinities to be assessed, but determination of the specific oligomeric species interacting with the lipids is not possible. Consequently, we have developed novel aggregation lipidbinding assays using SELDI-TOF MS, in which vesicles are adsorbed to hydrophobic H50 ProteinChip arrays, forming a supported lipid monolayer with the hydrophilic heads facing the solution. This assay, unlike conventional techniques, allows the rapid detection and quantitation of individual oligomeric species which are bound specifically and directly to the lipid-coated array. 2. Materials 2.1. Capture and Quantitation of Aβ Using Antibody-Coated ProteinChip Arrays 1. ProteinChip arrays: PS10, PS20 (Bio-Rad Laboratories, Hercules, CA). 2. Ab: Anti-human A� (0.5 mg/mL) in Tris-free buffer (see Table 1 for list of commonly used Abs). 3. A� peptide standards. 4. Phosphate-buffered saline (PBS): 137 mM NaCl, 10 mM phosphate buffer, pH 7.4. 5. Deactivation buffer: 0.5 M ethanolamine in PBS, pH 8.0. 6. PBST: PBS, 0.5% Triton X-100. 7. 1 mM hydroxyethyl piperazine ethane sulfonate (HEPES), pH 7.2. Table 1 Common Ab Used for Detection of A�, Including Information Pertaining to the Type (mAb/pAb) and Specificity of the Ab (epitope recognition site) Antibody Type (mAb/pAb) Specificity (Ref.) 4G8 mAb 17-24 aa (25) 6E10 mAb 1-12 aa (25) G210, G211 mAb C-terminal residues of A�-40 and -42, respectively (26) WO2 mAb 4-10 aa (27) Anti-A� 40 pAb 704-711 aa of APP (28) Anti-A� 42 pAb 706-713 aa of APP (28)
76 Giannakis et al. 8. Acetonitrile (ACN), HPLC grade. 9. Trifluoroacetic acid (TFA), HPLC grade. 10. Matrix/Energy Absorbing Molecule (EAM): �-cyano-4-hydroxy cinnamic acid (CHCA), 5 mg/tube (Bio-Rad Laboratories). 11. ProteinChip reader (Bio-Rad Laboratories). 12. Bioprocessor (optional) (Bio-Rad Laboratories). 2.2. Capture of Aβ Aggregates on ProteinChip Arrays 1. ProteinChip arrays: H50 (Bio-Rad Laboratories). 2. A� peptides. 3. Hexafluoroisopropanol. 4. 20 mM NaOH. 5. 10 mM phosphate buffer, pH 7.4 (PB). 6. 1 mM HEPES, pH 7.4. 7. 20 �M minisart RC4 filters (Sartorius, Göttingen, Germany). 8. ACN. 9. TFA. 10. Matrix/EAM: CHCA. 11. UV spectrophotometer. 12. ProteinChip reader. 2.3. Capture of Aβ on Lipid-Coated ProteinChip Arrays 1. ProteinChip arrays: H50. 2. 1-Palmitoyl-2-oeoyl-sn-glycero-3 [phospho-l-serine] (POPS) and 1 -palmitoyl- 2-oleoyl-sn-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids, Inc., Alabaster, AL). 3. A� peptides. 4. Ethanol-free chloroform. 5. Acid-washed glass beads, G1152 (Sigma-Aldrich, Poole, UK). 6. Liquid nitrogen. 7. 0.05 �M filters and spacers for extruder (Nuclepore Corp., Pleasanton, CA). 8. PB. 9. CHAPS (30 mg/mL). 10. 1 mM HEPES, pH 7.4. 11. ACN. 12. TFA. 13. Matrix/EAM: CHCA. 14. Sonicator. 15. Extruder (Avanti Polar Lipids). 16. ProteinChip reader.
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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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Page 38 and 39: 26 Bulet bioactive peptides from th
Page 40 and 41: 28 Bulet 21. Chernysh, S., Kim, S.I
Page 42 and 43: 3 Sequence Analysis of Antimicrobia
Page 44 and 45: Sequence Analysis of Antimicrobial
Page 58 and 59: 4 The Spot Technique: Synthesis and
Page 60 and 61: The Spot Technique 49 3. For amine
Page 62 and 63: The Spot Technique 51 2. Biotinylat
Page 64 and 65: The Spot Technique 53 the solutions
Page 66 and 67: The Spot Technique 55 solution. Ens
Page 68 and 69: The Spot Technique 57 (see Fig. 3)
Page 70 and 71: The Spot Technique 59 Fig. 5. Princ
Page 72 and 73: The Spot Technique 61 library, the
Page 74 and 75: The Spot Technique 63 7. Regenerati
Page 76 and 77: The Spot Technique 65 following rul
Page 78 and 79: The Spot Technique 67 20. Atherton,
Page 80 and 81: The Spot Technique 69 50. Bolger, G
Page 82 and 83: 5 Analysis of A� Interactions Usi
Page 84 and 85: Analysis of Aβ Interactions 73 are
Page 88 and 89: Analysis of Aβ Interactions 77 3.
Page 98 and 99: 6 NMR in Peptide Drug Development J
Page 100 and 101: NMR of Peptides 89 usually require
Page 102 and 103: NMR of Peptides 91 limiting the siz
Page 104 and 105: NMR of Peptides 93 and STD NMR expe
Page 106 and 107: NMR of Peptides 95 The basis of the
Page 108 and 109: NMR of Peptides 97 Fig. 5. Overlay
Page 110 and 111: NMR of Peptides 99 Fig. 7. Schemati
Page 112 and 113: NMR of Peptides 101 4.4.2. Saturati
Page 114 and 115: NMR of Peptides 103 4.6. Diffusion
Page 116 and 117: NMR of Peptides 105 Fig. 13. Propos
Page 118 and 119: NMR of Peptides 107 protein prevent
Page 120 and 121: NMR of Peptides 109 6. Wuthrich, K.
Page 122 and 123: NMR of Peptides 111 45. Morris, K.
Page 124 and 125: NMR of Peptides 113 78. Aumailley,
Page 126 and 127: 116 Copps et al. Fig. 1. Potential
Page 128 and 129: 118 Copps et al. Fig. 2. Flowchart
Page 130 and 131: 120 Copps et al. 10. In accordance
Page 132 and 133: 122 Copps et al. Fig. 3. DSSP for g
Page 134 and 135: 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
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Peptidomimetics 225 O O R S N H Pep
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Peptidomimetics 227 not without its
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Peptidomimetics 229 Oxidation step:
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Peptidomimetics 231 of peptidosulfo
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Peptidomimetics 233 8. Allow the re
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Peptidomimetics 235 3.3.2. Solid-Ph
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Peptidomimetics 237 On the other ha
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Peptidomimetics 239 nitrogen (73).
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Peptidomimetics 241 3. Cover the re
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Peptidomimetics 243 efficient synth
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Peptidomimetics 245 55. Alsina, J.,
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14 Synthesis of Toll-Like Receptor-
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Lipopeptides 249 2. Materials 2.1.
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Lipopeptides 251 Fig. 1. Structural
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Lipopeptides 253 8. To enable lipid
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Lipopeptides 255 Representative res
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Lipopeptides 257 Fig. 2. Immunogeni
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Lipopeptides 259 8. A large plastic
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Lipopeptides 261 26. Nardin, E.H.,
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264 Otvos response, synthetic pepti
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266 Otvos Fig. 3. Schematic present
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268 Otvos 3. Methods The main goal
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270 Otvos 3.2.3. Purification and Q
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272 Otvos 6. Meyer, D., and Torres,
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16 Cysteine-Containing Fusion Tag f
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Targeted Therapeutic and Imaging Ag
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Index A A� peptides aggregation a
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Index 297 phosphopeptides influenci
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Index 299 for genetically synthetic
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Index 301 peptidosulfonamide, disad
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Index 303 solid phase, 180, 214 sul
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Peptide-Based Drug Design

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