Peptide-Based Drug Design

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10 Bulet Key Words: Invertebrate immunity; antimicrobial peptides; immune effectors; mass spectrometry; drug discovery; Drosophila; arthropods; peptide purification; molecular mass fingerprints; bioactive peptides. 1. Introduction To date, three major classes of molecules have been developed for the treatment of human diseases. These types of molecules are small molecules, protein drugs, and peptides. Currently, most of the therapeutic molecules developed and marketed are small molecules. However, with the recent advancements in several technologies in the areas of (1) peptide manufacturing (chemical synthesis, recombinant expression), (2) screening, (3) stability, (4) modifications, and (5) improvements in peptide drug delivery systems, peptides are now considered as lead molecules for therapeutics (http://pubs.acs.org/cen/business/83/i11/8311bus1.html). Nature is an unlimited pipeline of bioactive peptides that are active regulators, information/signaling factors, and key components of the defense reactions, properties that make them interesting for drug discovery and development. Among the peptides, the bioactive peptides from the defense mechanisms of living organisms have such a therapeutic potential (1–4). Host-defense peptides are widely distributed in nature. Bacteria produce peptide antibiotics to mediate their microbial competitions (5,6). In multicellular organisms from both the vegetal and animal kingdoms, host-defense peptides such as antimicrobial peptides (AMPs) form a first line of host defense against pathogens (7–11). AMPs are critical effectors of the innate immunity of many species and are playing an essential role in terms of resistance to infection and survival. In living organisms, AMPs were found to be either constitutively stored or produced upon infection (natural or experimental). In invertebrates, for example, AMPs were found to be stored in immune competent blood cells (hemocytes) and in epithelial cells of a range of tissues. Interestingly, the presence of AMPs and other immune defense molecules in the hemolymph (the blood of invertebrates) or at the surface of the epithelia is only present if an infection is detected (12–17). AMPs were also reported to be constituents of the venom of arthropods (18). In addition to AMPs, a variety of other immune-induced molecules are produced to clear microbial infections; this was illustrated in the fruit fly model, Drosophila melanogaster (19–21). Therefore, together with other effectors of invertebrate immunity, they are considered potential candidates for their development as therapeutic drugs or as markers for diagnosis with applications in disease control. Such a distribution within body fluids (hemolymph but also venom) or immunocompetent cells, which is dependant of an immune state, is particularly important to consider
Bioactive <strong>Peptide</strong>s in Invertebrate Immune Systems 11 when collecting natural samples for the isolation of bioactive peptides playing a function in immune defenses. The purpose of this chapter is to provide a handbook for (1) induction of an immune state, (2) extraction, (3) purification, and (4) characterization (primary structure and biological properties) of immune-defense peptides (with a special reference to AMPs) from invertebrates. The methods detailed in this contribution are adapted to the extraction of immune factors from hemocytes, epithelial tissues, and hemolymph including small individuals such as Drosophila. Methods are largely based on those we used for the isolation and characterization of Drosophila immune-induced effectors (19,20). Methods reported in this contribution are also derived/updated/optimized from those reported for the identification and characterization of AMPs from noninsect arthropods and other invertebrates (22). Purification methods (liquid chromatography) and characterization (MALDI- and ESI-MS, MS/MS [tandem MS or MS n ], Edman sequencing, enzymatic digestions) strategies are based on the common physicochemical properties of defense peptides from the immune system, in particular on their relatively high hydrophobicity, cationic character at physiological pH, and short length (mostly below 10 kDa). Finally, structure determination techniques appropriate for the full characterization of immune factors are given together with a brief description of well-recognized antimicrobial assays (antibacterial and antifungal) when the immune effectors are members of the AMP armamentarium. 2. Materials 2.1. Animals 2.1.1. Unchallenged Animals Wild or bred laboratory individuals or animals from breeding farms at the same developmental stage (larvae or adults) with a similar size. 2.1.2. Experimentally Infected Individuals 1. Living or heat-killed bacteria: Gram-positive and Gram-negative ATCC strains. 2. Spores from a filamentous fungus (Pasteur Collection or private collections) or a yeast strain. 3. Lipopolysaccharides or peptidoglycans (Sigma, St. Louis, MO). 4. Infection with parasites: e.g., Crithidia species (23), Leishmania major (24). 5. Standard nutrient media for bacterial (Luria Broth Miller, Muller-Hinton Broth) and fungal growth (Sabouraud, yeast culture media).
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 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
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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
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Page 68 and 69: The Spot Technique 57 (see Fig. 3)
Page 70 and 71: 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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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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