Peptide-Based Drug Design

More documents

Recommendations

Info

The Spot Technique 61 library, the advantage of the random peptide array is that the peptides on each spot are unique, providing the possibility of higher individual activity. The disadvantage is in the lower number of synthesized peptides available for screening. 3.2.4.2. TRUNCATION ANALYSIS (LENGTH SCAN) To investigate the minimum possible length of an active peptide while maintaining activity, variations of the peptide sequence are synthesized by systematic shortening at the C-terminus, N-terminus, or both simultaneously (48,62,63). 3.2.4.3. LOOP SCAN (CYSTEINE SCAN) In order to stabilize loop structures or to increase their resistance to proteolytic digestion, it is convenient to cyclize peptides. There are two main types of cyclization: cyclization via cysteines forming a disulfide bridge and cyclization via a pair of peptide amino and carboxy groups to form an amide bond. In both cases, a pair of amino acids is involved. If they are not present in the peptide sequence, they must be inserted or two existing amino acids should be replaced, which could lead to a loss of activity. Therefore it is necessary to investigate the effect of cyclization on the activity of the peptide (64). For a cysteine loop scan, generate, synthesize, and test a set of all possible combinations of insertions or replacements using a pair of cysteines (see Fig. 6). For amide cyclization it is important that you have an amino group–containing amino acid such as lysine and a carboxy group–containing one like aspartic acid. That affords a higher number of possible combinations. A) B) 1.PEPTIDES 2.CCPTIDES 3.CECTIDES 4.CEPCIDES 5.CEPTCDES 6.CEPTICES 7.CEPTIDCS 8.CEPTIDEC 9.PCCTIDES 10.PCPCIDES 11.PCPTCDES 12.PCPTICES 13.PCPTIDCS 14.PCPTIDEC 15.PECCIDES 16.PECTCDES 17.PECTICES 18.PECTIDCS 19.PECTIDEC 20.PEPCCDES 21.PEPCICES 22.PEPCIDCS 23.PEPCIDEC 24.PEPTCCES 25.PEPTCDCS 26.PEPTCDEC 27.PEPTICCS 28.PEPTICEC 29.PEPTIDCC Fig. 6. Principle of a loop scan on the example of the peptide PEPTIDES.The amino acids were replaced by a pair of cysteines at all possible positions. (A) Image of the loop scan (fictive). (B) List of the sequences.
62 Winkler and Campbell 3.3. Probing The number of probing possibilities is huge and even includes testing the activity of peptides on living cells (14) or bacteria (35). Herewedescribethe use of labeled proteins such as antibodies. The labels described are horseradish peroxidase, alkaline phosphatase, and biotin. However, in general, most labeled proteins can be used according the method described in Subheading 3.3.1. Differences are in label-specific detection of the bound labeled protein. 3.3.1. HRP-Labeled Proteins and Chemiluminescence 1. If the membrane is dry, wash twice for 10 min each with methanol (or ethanol). Then wash three times with T-TBS or T-PBS for at least 5 min each. 2. Blocking: To decrease unspecific binding treatment with blocking buffer I or blocking buffer II is necessary (see Note 2). Blocking should be carried out for at least 2 h at room temperature. Treatment overnight at room temperature or over the weekend at 4 ◦ C is also possible. 3. Incubation with protein sample: Wash once with T-TBS or T-PBS for at least 5 min. After washing, incubate the membrane with the protein sample for at least 2 hat37 ◦ C. Treatment can also be carried out overnight at room temperature. 4. Incubation with a target protein binding protein (e.g., primary antibody): Wash three times with T-TBS or T-PBS for at least 5 min each. After washing, incubate the membrane with the (HRP-labeled) primary antibody solution for 2 h at room temperature or 37 ◦ C. 5. Incubation with secondary antibody directed against the protein used in step 4 (if no HRP-labeled primary antibody was used): Wash three times with T-TBS or T-PBS for at least 5 min each. After washing, incubate the membrane with the HRP-labeled secondary antibody solution for at least 1 h at room temperature or 37 ◦ C. 6. Detection: Wash at least three times for at least 5 min with TBS or PBS. Preparation of the chemiluminescence solution and detection (see Note 14): 22 �L of 80 mM p-coumaric acid in DMF and 50 �L of 250 mM luminol in DMF should be mixed with 1 mL 1 M Tris-HCl pH 8.5. Add to this mixture the corresponding amount of water (see Materials). The staining solution has to be activated by adding 3 �L of 30% H2O2 shortly before use. Excess washing buffer on the membrane has to be removed by placing the membrane on paper towels (see Note 15). Use forceps for all manipulations of the membrane. Never use fingers! The membrane then has to be placed on a plastic sheet (PP or PE). The detection buffer is poured over the whole membrane. To ensure homogeneous distribution the membrane has to be gently shaken on the plastic sheet. A reaction time of approximately 1 min is recommended. Detection of the chemiluminescence can be carried out using X-ray film or a chemiluminescence imager (62).
Page 2 and 3:
Peptide-Based Drug Design
Page 4 and 5:
METHODS IN MOLECULAR BIOLOGY TM Pep
Page 6 and 7:
Preface Natural products chemistry
Page 8 and 9:
Contents Preface...................
Page 10 and 11:
Contributors Nikolinka Antcheva •
Page 12 and 13:
Contributors xi Alessandro Tossi
Page 14 and 15:
2 Otvos advance of computer power a
Page 16 and 17:
4 Otvos see derivatives active in b
Page 18 and 19:
6 Otvos was designed based on ligan
Page 20 and 21:
8 Otvos 27. Borghouts, C., Kunz, C.
Page 22 and 23: 10 Bulet Key Words: Invertebrate im
Page 24 and 25: 12 Bulet 6. 5 �L or higher volume
Page 26 and 27: 14 Bulet 2.5.2.1. MALDI-TOF-MS 1. M
Page 28 and 29: 16 Bulet 7. Small-volume low-protei
Page 30 and 31: 18 Bulet 3. Centrifuge between 8000
Page 32 and 33: 20 Bulet internal diameter). Increa
Page 34 and 35: 22 Bulet interest. As no instrument
Page 36 and 37: 24 Bulet 3. Incubate the plates in
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 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
Page 142 and 143:
132 Hilpert et al. different natura
Page 144 and 145:
134 Hilpert et al. wt A C D E F …
Page 146 and 147:
136 Hilpert et al. methods primaril
Page 148 and 149:
Page 150 and 151:
Page 152 and 153:
Page 154 and 155:
144 Hilpert et al. Table 3 Summary
Page 156 and 157:
Page 158 and 159:
148 Hilpert et al. of substituting
Page 160 and 161:
150 Hilpert et al. in models that c
Page 162 and 163:
152 Hilpert et al. than control. Gi
Page 164 and 165:
154 Hilpert et al. Table 4 Accuracy
Page 166 and 167:
156 Hilpert et al. 25. Pini, A., Gi
Page 168 and 169:
158 Hilpert et al. 55. Giacometti,
Page 170 and 171:
9 Investigating the Mode of Action
Page 172 and 173:
Proline-Rich Antimicrobial Peptides
Page 174 and 175:
Page 176 and 177:
Page 178 and 179:
Page 180 and 181:
Page 182 and 183:
Page 184 and 185:
Page 186 and 187:
10 Serum Stability of Peptides Håv
Page 188 and 189:
Serum Stability of Peptides 179 2.
Page 190 and 191:
Page 192 and 193:
Serum Stability of Peptides 183 �
Page 194 and 195:
Page 196 and 197:
11 Preparation of Glycosylated Amin
Page 198 and 199:
Glycosylated Amino Acid Synthesis 1
Page 200 and 201:
Page 202 and 203:
Page 204 and 205:
Page 206 and 207:
Page 208 and 209:
Page 210 and 211:
Page 212 and 213:
Page 214 and 215:
Page 216 and 217:
Page 218 and 219:
12 Synthesis of O-Phosphopeptides o
Page 220 and 221:
Solid-Phase Synthesis of O-Phosphop
Page 222 and 223:
Page 224 and 225:
Page 226 and 227:
Page 228 and 229:
Page 230 and 231:
Page 232 and 233:
13 Peptidomimetics: Fmoc Solid-Phas
Page 234 and 235:
Peptidomimetics 225 O O R S N H Pep
Page 236 and 237:
Peptidomimetics 227 not without its
Page 238 and 239:
Peptidomimetics 229 Oxidation step:
Page 240 and 241:
Peptidomimetics 231 of peptidosulfo
Page 242 and 243:
Peptidomimetics 233 8. Allow the re
Page 244 and 245:
Peptidomimetics 235 3.3.2. Solid-Ph
Page 246 and 247:
Peptidomimetics 237 On the other ha
Page 248 and 249:
Peptidomimetics 239 nitrogen (73).
Page 250 and 251:
Peptidomimetics 241 3. Cover the re
Page 252 and 253:
Peptidomimetics 243 efficient synth
Page 254 and 255:
Peptidomimetics 245 55. Alsina, J.,
Page 256 and 257:
14 Synthesis of Toll-Like Receptor-
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
Page 264 and 265:
Lipopeptides 255 Representative res
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
Page 280 and 281:
272 Otvos 6. Meyer, D., and Torres,
Page 282 and 283:
16 Cysteine-Containing Fusion Tag f
Page 284 and 285:
Targeted Therapeutic and Imaging Ag
Page 286 and 287:
Page 288 and 289:
Page 290 and 291:
Page 292 and 293:
Page 294 and 295:
Page 296 and 297:
Page 298 and 299:
Page 300 and 301:
Page 302 and 303:
Index A A� peptides aggregation a
Page 304 and 305:
Index 297 phosphopeptides influenci
Page 306 and 307:
Index 299 for genetically synthetic
Page 308 and 309:
Index 301 peptidosulfonamide, disad
Page 310:
Index 303 solid phase, 180, 214 sul
show all

Peptide-Based Drug Design

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?