Peptide-Based Drug Design

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Lipopeptides 249 2. Materials 2.1. Synthesis of Lipopeptides 1. Solid phase supports are from Rapp Polymere GmbH (Germany). The substitution levels of supports used in this laboratory are in the range 0.15–0.4 mmol/g. 2. Fluorenylmethoxycarbonyl (Fmoc) derivatives of amino acids (obtained from Auspep, Australia or Merck, Germany). 3. Solvents and reagents used should be of analytical grade or equivalent: a. N,N´-Dimethylformamide (DMF; Auspep, Australia or Merck, Germany). b. Dichloromethane (DCM; Merck, Australia). c. Diethylether (Merck, Germany). d. Diisopropylethylamine (DIPEA; Sigma-Aldrich, Australia). e. 1,8-Diazabicyclo-[5.4.0]undec-7-ene (DBU; Sigma-Aldrich, Germany). f. O-benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate (HBTU; Merck, Germany). g. 1-Hydroxybenzotriazole (HOBT; Auspep, Australia). h. 2,4,6-Trinitrobenzenesulfonic acid (TNBSA; Fluka Biochemika, Switzerland). 4. Pam2Cys attachment to peptides: a. Diisopropylcarbodiimide (DICI; Auspep, Australia). b. Palmitic acid (Merck, Germany) . c. 4-[Dimethylamino]-pyridine (DMAP; Fluka Biochemika, Switzerland). d. 3-Bromo-1,2-propanediol (Fluka Biochemika, Switzerland). e. Fluorenylmethoxylcarbonyl-N-hydroxysuccinimide(Novabiochem, Germany). f. Triethylamine (Merck, Germany). g. l-Cysteine hydrochloride monohydrate (Sigma-Aldrich, USA). 5. Cleavage of lipopeptide from resin: a. Phenol (Sigma-Aldrich, USA). b. Triisopropylsilane (TIPS; Sigma-Aldrich, Australia). c. Trifluoroacetic acid (TFA; Auspep, Australia). 6. Purity analysis and characterization of synthesized lipopeptide: a. Acetonitrile (Merck, Germany). b. High-performance liquid chromatography (HPLC) system (Waters, Australia). c. C4 column (4.6 × 300 mm; Vydac, USA). d. Ion-trap mass spectrometer (Agilent 1100 Series LC/MSD) (Agilent Technologies, Australia). 2.2. Induction of Antibody Response in Mice 2.2.1. Inoculation of Mice 1. 6- to 8-Week-old female mice (BALB/c or C57BL6), 5 mice per group. 2. Saline.
250 Chua et al. 3. Complete Freund’s adjuvant (CFA; Sigma-Aldrich, USA). 4. Incomplete Freund’s adjuvant (IFA; Sigma-Aldrich, USA). 2.2.2. Enzyme-Linked Immunosorbent Assay 1. Flat-bottom well polyvinyl plates (Thermo, USA). 2. PBSN3, phosphate-buffered saline (PBS) containing v/v 0.1% sodium azide. 3. LHRH peptide (HWYSGLRPG) dissolved at 5 �g/mL in PBSN3. 4. PBST, PBS containing v/v 0.05% Tween-20 (Sigma-Aldrich, USA). 5. BSA10PBS, PBS containing 10 mg/mL bovine serum albumin (Sigma-Aldrich, USA). 6. BSA5PBST, PBST containing 5 mg/mL bovine serum albumin (Sigma-Aldrich, USA). 7. Horseradish peroxidase-conjugated rabbit anti-mouse IgG antibodies (Dako, Denmark). 8. Enzyme substrate: 0.2 mM 2,2´-azino-bis 3-ethylbenzthiazoline-sulfonic acid (ABTS; Sigma-Aldrich, USA) in 0.05 M citric acid (M&B, England) containing 0.004% hydrogen peroxide(Ajax Chemicals, Australia). 9. 0.05 M Sodium fluoride (NaF; BDH Chemicals, Australia). 10. ELISA plate reader (Labsystems Multiskan, USA). 3. Methods 3.1. Synthesis of Lipopeptide The lipopeptide vaccine described in this study consists of a CD4 + helper T-cell epitope ([T]) and a B-cell epitope ([B]). These two epitopes are separated by a lysine residue (K) to which is attached the lipid moiety via two serine residues (Fig. 1B). TheCD4 + T-helper epitope KLIPNASLIENCTKAEL used is derived from the fusion protein of the morbillivirus canine distemper virus (34) and is recognized by T cells from BALB/c and C57BL6 mouse strains (4). The B-cell epitope is LHRH and has the sequence HWYSGLRPG. The presence of anti-LHRH antibodies can render vaccinated animals sterile. We have found that although the use of an automatic synthesizer can save time and can be relatively unproblematic for simple sequences, the synthesis of peptides manually allows for more flexibility and control over the assembly process. This is particularly important for the synthesis of difficult sequences as it permits quick and easy intervention at any point. The apparatus routinely used in this laboratory for the manual synthesis of peptides consists of a flask attached to a glass manifold that can support up to four sintered funnels, thereby permitting the simultaneous synthesis of up to four peptides. The side arm of the flask is attached to a vacuum pump to allow for solvents to be aspirated from each funnel. The manifold also contains valves
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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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Page 208 and 209: Glycosylated Amino Acid Synthesis 1
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Page 220 and 221: Solid-Phase Synthesis of O-Phosphop
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Page 234 and 235: Peptidomimetics 225 O O R S N H Pep
Page 236 and 237: Peptidomimetics 227 not without its
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Page 240 and 241: Peptidomimetics 231 of peptidosulfo
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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 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 302 and 303: Index A A� peptides aggregation a
Page 304 and 305: Index 297 phosphopeptides influenci
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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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