Peptide-Based Drug Design

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24 Bulet 3. Incubate the plates in the dark for 48 h in a moist chamber at 25 ◦ C. 4. Measure fungal growth by detecting culture absorbency in a microplate reader (600 nm). A control of the growth can be performed after 24 h using a photonic microscope. 4. Notes 1. If the solution is yellowish discard it. 2. To kill E. coli D31, ciprofloxacin, which belong to the group of fluroquinolones, may be used as a positive control antibiotic. 3. Experimental infections, tissue collection, extraction conditions, purification procedures, and strategies for determining the primary structure of the natural bioactive peptides should be adapted to the animal model (size, rarity, possibility to conduct molecular biology investigations, availability of EST, and genomic databases, etc.) considered and to the complexity of the bioactive peptide. 4. Different microorganisms or parasites are used as inducers of the immune response of invertebrates. Constituents of the bacterial membrane may also be considered as successful inducers. Nevertheless, they may not develop the full immune response, limiting the possibility to recover all the armamentarium build by the invertebrate to fight off an infection (34). 5. A relevant set of not experimentally infected individuals (control) is required for differential analysis by MALDI-TOF-MS or RP-HPLC in order to discriminate between immune-induced molecules and constitutively present substances (19). This is a prerequisite when no in vitro assays are used to select the bioactive peptides from the immune system of the model invertebrate investigated. 6. Working on an animal model with a limited volume of hemolymph (
Bioactive <strong>Peptide</strong>s in Invertebrate Immune Systems 25 120 experimentally infected flies) was loaded onto a microbore RP column (Aquapore RP 300 C8, 1× 100 mm, Brownlee). Elution was performed either with a linear gradient of 0–80% acetonitrile in acidified water over 80 min (19,27) or with a linear gradient of 2–62% acetonitrile in acidified water over 60 min (30), both at a flow rate of 80 �L/min at 35 ◦ C. 11. If the molecule of interest is eluting during the first HPLC run with the front, the following purification step should be performed in an isocratic condition with a minimum of acetonitrile (0.5%) or directly in acidified water without acetonitrile. 12. Using RP-HPLC, the alkylated peptide is separated from reagents and the unreacted peptide with a gradient of acidified acetonitrile from 2 to 80% in 120 min. The large quantity of reagents is eliminated owing to long washing period (at least 30 min) with 2% acidified acetonitrile prior starting the gradient. Using the 4-vinylpyridine, an increase in the absorbance is observed on the alkylated peptide compared to the native molecule. When using magnetic beads, the chemically modified peptide is recovered by elution with 60% acetonitrile. 13. For molecular mass fingerprint of an enzymatic digestion, the acidity of the matrix and of the 1% TFA droplet is sufficient to quench any further digestion. 14. For determining the molecular mass of a polypeptide, a single-stage mass spectrometer is appropriate, whereas for analysis of structural features tandem MS (MS n ) is required. In this latter case, after molecular mass measurement, specific ions are selected and then subjected to fragmentation through collision in a specific chamber (collision cell) supplied with an appropriate gas (e.g., argon). Instrument performance (resolution, sensitivity, mass accuracy) depends on the instrument type, the ionization method, and the scanning capabilities. 15. For the analysis of a trypsin digest by MS, an internal calibration is performed with fragments of autolysed trypsin (e.g., [M-H] + at m/z 842.510, 1045.546, and 2211.104). 16. To avoid the inconvenience of the TFA (decrease sensitivity) during MS, if molecular mass measurements are performed on fractions that may contain traces of TFA, the sample has to be desalted and concentrated onto a C18 Zip-Tip. Elution of the peptide(s) will then be performed by 50% aqueous acetonitrile supplemented with 1% formic acid. 5. Conclusion Intensive research efforts for developing new anti-infectious and antitumoral drugs for human health rely on technological advancements in screening of combinatorial chemical libraries and/or natural extracts generated from tissues and fluids from animals and plants. Presently, the use of natural extracts and the random HTS of libraries of synthetic or natural molecules have successively allowed the discovery of new therapeutic molecules. The race to discover the new drugs of tomorrow has begun. In the area of antibiotics, facing the increased resistance of bacteria and fungi to conventional antibiotics, the natural
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 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
Page 82 and 83: 5 Analysis of A� Interactions Usi
Page 84 and 85: 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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