Peptide-Based Drug Design

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20 Bulet internal diameter). Increasing the flow rate will give a better resolution but will also generate higher elution volumes. 3. Fractions are collected manually according to the absorbance measured either at 214 nm (increased sensitivity, higher background) or at 225 nm (best signal/solvent ratio). Using this procedure, each fraction corresponds to an individual absorbance peak. This minimizes the number of steps to get pure material for structural analysis. 4. Fractions are analyzed by MS or subjected to in vitro assays. For MS analysis, the collected fractions can be directly analyzed following dilution in the appropriate solvent. For activity screening, fractions are dried in a vacuum-centrifuge, reconstituted either in water or in a suitable buffer adapted to the activity monitoring. 3.3.2.2. STEP 2: RP-HPLC OR SIZE EXCLUSION HPLC Depending of the results of MS, step 2 can be performed either by RP-HPLC or by SEC. For RP-HPLC, the same column as in step 1 can be used, but the fractions are eluted using linear biphasic gradients of acetonitrile in acidified water at a same flow rate and at the same temperature (an increase in the temperature would result in a better resolution). The gradient is chosen according to the following strategy: from 2% to ([C]-2)% acetonitrile in 10 min and from ([C]-2)% to ([C]+15)% acetonitrile at an increase concentration of 0.2–0.3%, where [C] corresponds to the exact concentration of acetonitrile for the elution of the compound of interest (see Note 11). SEC is preferred when MS measurements reveal the presence of molecules with molecular masses rather distant (e.g., >10 kDa and 2–4 kDa). 1. Inject the fraction of interest (biologically active, immune induced) on two serially linked size exclusion columns (equivalent to the former Beckman SEC 3000 and SEC 2000 columns, 300 mm × 7.5 mm or any other brand). Use of a precolumn is recommended to protect expensive columns. The injected volume should not exceed 100 �L for adequate resolution. 2. Elute with 30% acetonitrile in acidified water at a flow rate below 0.5 mL/min. Fractions are collected manually according to the absorbance at 214 nm. Using SEC, detection at 214 nm is not a problem as isocratic conditions are used. 3. For the procedure, see Subheading 3.3.2.1., step 4. 3.3.2.3. FINAL PURIFICATION STEP: RP-HPLC The final purification step is a linear biphasic gradient of acidified acetonitrile at a flow rate between 0.08 and 0.25 mL/min (0.08 mL for the microbore column, 0.1–0.25 mL for the narrowbore column). The following two-step gradient for step 2 is used. However, to increase resolution, a column with a smaller internal diameter is needed and column temperature is increased to 37◦C. For fraction handling, use the procedure according to Subheading 3.3.2.1., part 4.
Bioactive <strong>Peptide</strong>s in Invertebrate Immune Systems 21 3.4. Reduction and Alkylation 1. Dissolve or dilute the peptide (few to 100 pmol) in an Eppendorf tube using the reaction buffer. 2. Add 2 �L of the reducing agent in a large excess (e.g., 2.2 M DTT or TCEP). 3. Flush the tube containing the sample with N2 to prevent oxidation and incubate at 45 ◦ Cfor1h. 4. Add 2 �L of the alkylated agent: pure colorless 4-VP or 2.5% (w/v) iodoacetamide for alkylation of the peptide and of the residual reducing agent. Flush again the Eppendorf tube with N2 and incubate for 10 min in the dark. 5. Stop the reaction by desalting the reaction mixture by SPE onto RP-cartridges, by RP-HPLC or SEC, or by functionalized magnetic beads (see Note 12). 3.5. Enzymatic Cleavages 1. Reconstitute the dried peptide in the digestion buffer corresponding to the enzyme of interest in the conditions recommended by the manufacturer. 2. Incubate at the appropriate temperature (37 ◦ C is often recommended) for 1 to 16 h depending on the time-course properties of the enzyme. For the C-terminal sequencing by carboxypeptidase P digestion, a time course digestion is suitable (e.g., 30 s, 2 min, 5 min, 10 min, etc. up to 48 h). 3. Stop the reaction by addition of 1 volume of an acidic solution (e.g., 0.1% TFA) and control the pH using pH paper (pH < 4). For direct molecular mass fingerprint see Note 13. 4. Perform immediately the purification of the digest mixture on a reversed-phase column or by fast desalting by micro SPE using a C18 Zip-Tip (Millipore). 3.6. Microsequencing by Edman Chemistry The following procedure may be used for native and reduced/alkylated peptides as well as for enzymatic peptide fragments. 1. Reconstitute the peptide in less than 10–15 �L with 30% acetonitrile in 0.1% TFA. Adapt the quantity to sequence according to the sequencer, to the molecular mass of the sample to analyze and to the number of amino acids to sequence (e.g., 50–100 pmol for a Procise System,
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 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 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
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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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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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