Peptide-Based Drug Design

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Analysis of Aβ Interactions 81 5. Filter the solution to ensure preformed aggregates are removed (20 �m Minisart RC4, Sartorius). 6. To account for the loss of peptide resulting from the filtering process, it is necessary to reassess the protein concentration by UV spectrophotometer. The extinction coefficient for A� fragments is determined by measuring light absorbance of A� dissolved in a high-pH solution (e.g., NaOH at pH 11 to allow for complete solubility). 7. Adjust samples to 10 �M and incubate at 37 ◦ C with shaking (200 rpm) to induce aggregation. 3.2.2. ProteinChip Analysis 1. Place H50 ProteinChip arrays in a humidity chamber. 2. Pre-equilibrate the arrays in the sample binding buffer; load each spot with 5 �L of PB and incubate for 5 min on a shaking table. Repeat for a total of two PB washes. 3. Adjust samples to 10 �M in PB and load 5 �L per spot. 4. Incubate for 2 h at room temperature on a shaking platform. 5. To remove nonspecifically bound peptide, wash spots three times with 5 �LofPB for 5 min on a shaking platform. 6. Finally wash arrays twice with 1 mM HEPES, pH 7.4 for 1 min. 7. Dry arrays and load 1 �L of 50% CHCA. Allow the array to air-dry and repeat the CHCA application. 8. Collect data at the low and high mass range. 9. Export data into an Excel spread sheet and determine the signal-to-noise/area under the curve for each oligomeric species (see Note 11). 3.3. Capture of Aβ on Lipid-Coated ProteinChip Arrays 3.3.1. Vesicle Preparation 1. Combine equal amounts of 1-Palmitoyl-2-oeoyl-sn-glycero-3 [phospho-l-serine] (POPS) and 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (16 mg) in a 10-mL glass conical flask and dissolve in 2 mL of ethanol-free chloroform. 2. Allow chloroform to evaporate for 1 h under a stream of nitrogen; this will create a thin layer of lipid in the bottom of the flask. 3. Add acid-washed glass beads and 1 mL of PB to the flask and incubate for 1 h at 37 ◦ C with agitation (220 rpm) to resuspend the lipid layer. 4. Transfer lipid to an Eppendorf tube, sonicate for 15 min, and subject sample to several freeze–thaw cycles (5×) using liquid nitrogen and a 37 ◦ C water bath. 5. Prepare the extruder apparatus according to the manufacturer’s specifications. 6. Place apparatus on a 37 ◦ C heating block. Pass the lipid through a 0.05 �M presoaked filter 11 times, after which time the solution will appear more translucent. 7. Dispense the solution into a glass jar and store under nitrogen at 4 ◦ C; use within 48 h.
82 Giannakis et al. 3.3.2. Formation of a Lipid Monolayer on H50 ProteinChip Arrays 1. Place H50 ProteinChip arrays in a humidity chamber. 2. Load each spot with 5 �L of CHAPS (30 mg/mL), and immediately wick off using aKimwipe. 3. Remove any residual detergent by washing with PB; load 5 �L of PB per spot and incubate for 2 min on a shaking platform; repeat for a total of three washes. 4. Load 5 �L of the 41 �M POPC/POPS mixture or PB to each spot and incubate for 2hat37◦C. 5. To remove unbound lipid, wash spots with 5 �LofPBtwicefor2minonashaking platform. 3.3.3. Protein Binding to the Lipid-Coated H50 ProteinChip Arrays 1. Prepare samples in PB to a final concentration of 50 �M andload5�L onto spots coated with either the POPC/POPS lipid mixture or PB alone. Fig. 3. Analysis of peptide–lipid interactions using ProteinChip arrays. Vesicles are absorbed to H50 ProteinChip arrays via interactions with C8 functional groups, creating a supported lipid monolayer on the chip surface (A, B). Samples are applied to the chip, incubated for 5 min (C), and washed to remove nonspecifically bound proteins (D). Matrix is added (E), and the array is introduced into the ProteinChip reader, where the laser is fired onto the chip surface. <strong>Peptide</strong>s retained on the surface are finally resolved by TOF-MS, displaying mass-to-charge versus signal intensity (F, G). (Adapted from ProteinChip technology training course, Bio-Rad Laboratories.)
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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
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
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Page 64 and 65: The Spot Technique 53 the solutions
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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 86 and 87: Analysis of Aβ Interactions 75 Ana
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
Page 136 and 137: 126 Copps et al. 19. Essman, U., Pe
Page 138 and 139: 128 Hilpert et al. Key Words: Scree
Page 140 and 141: 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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138 Hilpert et al. Table 2 (Continu
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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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