Peptide-Based Drug Design

More documents

Recommendations

Info

Serum Stability of Peptides 179 2. Cleaving reagent trifluoroacetic acid (TFA; Shanghai Fluoride Chemicals, Shanghai, China). 3. Acetonitrile (PerSeptive Biosystems, Framingham, MA), also known by the synonyms methyl cyanide, cyanomethane, ethanenitrile, and ethyl nitrile, is rather toxic and precautions should be taken. 4. Automatic peptide synthesizer, Milligen 9050 PepSynthesizer (Milligen, Milford, MA). 5. RP-HPLC (Waters Corporation, Milford, MA). 6. Electron-spray interface VG QUATTRO quadruple mass spectrometer (VG Instruments Inc., Altrincham, UK). 2.2. In Vitro Peptide Stability in Serum/Reaction Kinetics 1. RPMI medium 1640 (Gibco, Invitrogen, Carlsbad, CA) supplemented with 25% (v/v) of human serum (Fisher BioReagents, Fisher Scientific, Pittsburg, PA) (see Note 2). 2. Peptide stock solution: 1–10 mg/mL dissolved in pure dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO). 3. Trichloroacetic acid (TCA; Sigma, St. Louis, MO) (aq) 6% or 15% (w/v), or 96% ethanol. 4. TFA (Shanghai Fluoride Chemicals, Shanghai, China). 5. RP-HPLC buffer A: 0.08% TFA in water, buffer B: 0.08% TFA in acetonitrile. 2.3. In Vivo Peptide Stability Assay/Pharmacokinetics in Mice 1. Peptide dissolved in sterile phosphate-buffered saline (PBS) pH 7.2. 2. Balb/c mice (Charles River Laboratories, Wilmington, MA). 3. TCA (Sigma, St. Louis, MO) (aq) 15% (w/v). 3. Methods Today peptide synthesis is a mainstream operation in many big laboratories. Several companies have in addition specialized in providing custom-ordered peptides, e.g., large-scale peptides (made in solution), peptide libraries (made on cellulose membranes), or most commonly small peptide quantities (made on resin, solid phase synthesis). Although peptides are considered rather stabile in many test assays, concerns have been raised regarding their stability in presence of human serum. In a general serum stability assay, the peptide is subjected to human serum (see Note 3) at realistic temperature conditions and incubated for various time intervals, comparable to traditional protease assays. The reactions are stopped by TCA or ethanol, precipitating larger serum proteins while leaving peptides
180 Jenssen and Aspmo and peptide derivatives soluble for further analysis. To obtain reliable and reproducible results, peptide degradation is performed under conditions where the peptide concentration is not the rate-limiting concentration, but rather where the reaction speed is linearly dependent on the serum concentration (e.g., at 25% serum), i.e., the enzyme concentration is rate limiting, not the substrate concentration. Peptide analysis is performed on the supernatant with either mass spectrometry, e.g., MALDI-TOF (15), or with RP-HPLC under stability-specific chromatography conditions. It should be noted that mass spectrometry is rarely a valid quantitative measure without utilizing isotopic internal standards, while RP-HPLC analysis of peptides is directly quantitative with a UV detector. However, mass spectrometry of the degradation products will provide insight into if or where the peptide is cleaved and/or if other modifications to the peptide have occurred in the serum (glycosylations, phosporylations, or deglycosylations, dephosphorylations, etc.). It should also be taken into account that several factors may cause misleading stability results for the peptides (see Note 4). In vivo testing of peptide stability is obviously of more relevance than in vitro testing. However, a better term would probably be peptide pharmacokinetics (in mice), given that these measurements basically are also done in vitro. 3.1. Solid Phase Peptide Synthesis and Purification 1. Solid phase peptide synthesis has been described earlier in great detail (16). But, in brief, a standard way of doing this is by using Fmoc-chemistry (9-fluorenylmethoxycarbonyl), a baselabile protection group that easily can be removed during peptide chain elongation. 2. The peptides can be synthesized automatically using Fmoc amino acids and PAL- PEG resin on a Milligen 9050 PepSynthesizer (17). 3. The first amino acid is coupled onto the resin in the presence of DIPEA, using HBTU as a coupling reagent. 4. The next amino acid in the sequence is coupled to the active N-terminal end of the first amino acid, and the process can be repeated over and over until the entire peptide is made. 5. When the peptide is finished it can be cleaved from the solid support (resin) using TFA. This acid-based cleavage process also results in deprotection of the amino acid side chains. 6. The peptide is then precipitated out in ether and washed extensively with additional ether prior to drying. 7. The dried peptide is then weighed out and dissolved in water containing 1% acetonitrile, to a concentration of 20 mg/mL of peptide. 8. The peptides are purified by preparative RP-HPLC on a C18 column with a loading capacity of 20 mg.
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 46 and 47:
Page 48 and 49:
Page 50 and 51:
Page 52 and 53:
Page 54 and 55:
Page 56 and 57:
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 86 and 87:
Analysis of Aβ Interactions 75 Ana
Page 88 and 89:
Analysis of Aβ Interactions 77 3.
Page 90 and 91:
Page 92 and 93:
Page 94 and 95:
Page 96 and 97:
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 154 and 155: 144 Hilpert et al. Table 3 Summary
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 186 and 187: 10 Serum Stability of Peptides Håv
Page 190 and 191: Serum Stability of Peptides 181 9.
Page 192 and 193: Serum Stability of Peptides 183 �
Page 194 and 195: Serum Stability of Peptides 185 13.
Page 196 and 197: 11 Preparation of Glycosylated Amin
Page 198 and 199: Glycosylated Amino Acid Synthesis 1
Page 218 and 219: 12 Synthesis of O-Phosphopeptides o
Page 220 and 221: Solid-Phase Synthesis of O-Phosphop
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

Create successful ePaper yourself

Delete template?

Save as template?