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Design & Use of Antimicrobial Peptides

Design & Use of Antimicrobial Peptides. 3 . Current obstacles. 1. Mechanisms of action. 2. Synthesis strategies. 4. Future directions. Al Shaibani, MICB404 Feb 4, 2014. Cationic Host Defense Peptides. Suppress biofilm formation/accumulation Inhibition of cell wall biosynthesis

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Design & Use of Antimicrobial Peptides

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  1. Design & Use of Antimicrobial Peptides 3. Current obstacles 1. Mechanisms of action 2. Synthesis strategies 4. Future directions Al Shaibani, MICB404 Feb 4, 2014

  2. Cationic Host Defense Peptides • Suppress biofilm formation/accumulation • Inhibition of cell wall biosynthesis • Disruption of membrane integrity • Chemoattractants of phagocytes • Regulate innate immunity • Enhance wound healing & angiogenesis • Pro & anti-apoptotic effects • Adjuvants Anti-fungal, anti-parasitic activity? Hancock et. al. 2006

  3. Disturbs many biological functions with modest potency • Co-evolution and microbial counter-strategies = diverse shapes and targets • Natural cationic host defense peptides are attenuated by physiological conditions

  4. Selectivity of host defense peptide activity: • Cationic peptides bind the negatively charged lipid head groups then insert • Alter membrane structure • Favourmembranes with anionic lipids (cardiolipin, PG, etc.), high electric potential gradient, lack cholesterol • Modeled by Surface Plasmon Resonance

  5. 1. Mechanism of action Only a few strongly antimicrobial peptides: porcine protegrin & horseshoe crab polyphemusin I (β-hairpin peptides) Ex: Polyphemusin I • Inhibits growth of Gram –ve & Gram +ve bacteria • MIC is 0.5 – 1 ul • Does not have antimicrobial activity against P. aeruginosa • Contains hemolytic activity at high concentrations

  6. 1. Mechanism of action Ex: Plectasin • Defensin from saprophytic fungus • Secondary and tertiary structures resemble defensins in spiders, mussels, scorpions & dragonflies • Active against Streptococcus pneumonia • Targets bacterial cell wall precursor Lipid II

  7. Kinetics of bacterial killing in vitro. S. pneumoniae serotypes 2 • Comparative clearance of S. pneumonieaein vivo by Plectasin and Penicillin. • Pull down assay of Plectasin and Lipid II confirms interaction

  8. Peptide focus: PlectasinPlectasin inhibits cell wall biosynthesis

  9. Design & Use of Antimicrobial Peptides 4. Future directions 3. Current obstacles 1. Mechanisms of action 2. Synthesis strategies

  10. 2. Synthesis strategies • Activity is based on residue composition, size, overall charge, 2˚ structure, etc. • Challenge: find or make potent, safe, cheap and easily made peptides • QSAR approach

  11. A: Modification of known peptides • Enhance infective activity or decrease therapeutic toxicity based on alterations to amino acids. • Single mutations shed light on activity significance • “Local” approach as opposed to “global” approach • High throughput peptide synthesis on arrays + high-throughput & rapid luminescence-based assay of bacterial killing Linguistic model: “My ribcage and lizard are lean, and dentists that I work with may be beer-stained.”

  12. B: Rigorous biophysical modeling • Understand peptide activity in hydrophobic environments • Reshape peptides at atomic level (∆G free energy calculations) • Statistical dynamics & simulations • Uses indolicidin & bactenicin as templates for structure Ovispirin analogue

  13. C: Virtual Screening • Numerical methods to determine quantifiable peptide descriptors • Relate properties to bioactivity • Input = peptide properties, output = bioactivity • Stochastic optimization for peptides with “memory” AMPer Things to take into account: Isoelectric points NMR spectroscopy pH Molecular weight Van der Waal’s surface area … Methionine Cysteine

  14. Design & Use of Antimicrobial Peptides 4. Future directions 3. Current obstacles 1. Mechanisms of action 2. Synthesis strategies

  15. 3. Current obstacles • Many host defense peptides have NLS (ex: LL-37) • $100 – 600 per g of peptide! Current and proposed solutions: • New peptide production platforms • Robotically spot-synthesized peptides on cellulose sheet • Coupled with ATP-dependent Luciferase-expressing luminescence of P. aeruginosa • Make short peptides • Use D-amino acids • Chemical modifications (ex: long acyl chains, improving hydrophilicity, fluorousaa, etc.)

  16. Peptidomimetics

  17. Peptidomimetics • Mimicking drugs to make stable, selective peptides • “foldamers” – short & sequence specific oligomers • Versatile, 3D scaffold, based on computer simulations • Urea & Arylamide & ß-peptides backbones -> must contain amphiphilicity Phenylene-ethynylene AMP

  18. Design & Use of Antimicrobial Peptides 4. Future directions 3. Current obstacles 1. Mechanisms of action 2. Synthesis strategies

  19. 4. Future directions • Several synthetic peptides are in clinical trials now • Pexiganan passed phase III • Omiganan – proven to reduce catheter colonization • Peptidomimetics • Aerosolized formulations to treat CF • Using host defense peptides to regulate innate immune system? • Replacing antibiotics?

  20. Key points • Peptides lead to membrane/cell rupture, inhibit cell wall biosynthesis, macromolecular synthesis or metabolic functions. • Host defense peptides because they have direct antimicrobial activity and they enhance innate immune responses. • Diverse bioactivity and structure • Mechanism of action for many peptides is still unclear • Objective is to optimize peptide use and production • Peptidomimetics, language models and simulations are helping us understand host defense peptides!

  21. References Mygind, PH. et al. 2005. Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature. 437. 975-980. Schneider T. et al. 2010. Plectasin, a Fungal Defensin, Targets the Bacterial Cell Wall Precursor Lipid II. Science.328. 1168-1172. Zhang L, et al. 2005. Antimicrobial peptide therapeutics for cystic fibrosis. Antimicrob. Agents Chemother. 49:2921–2927. Khandelia H and Kaznessis YN. 2005. Molecular dynamics simulations of the helical antimicrobial peptide Ovispirin-1 in zwitterionicdodecylphosphocholine micelle: insights into host-cell toxicity. J. Phys. Chem. 109. 12990-12996. Pundir P, Catalli A, Leggiadro C, Douglas SE and Kulka M. 2014. Pleurocidin, a novel antimicrobial peptide, induces human mast cell activation through the FPRL1 receptor. Mucosal Immunology. 7:177-187. Hilpert K, Volkmer-Engert R, Walter T, Hancock REW. 2005. High-throughput generation of small antibacterial peptides with improved activity. Nature. 23:1008-1012. Wang Y, Chi EY, Schanze KS, Whitten DG. 2012. Membrane activity of antimicrobial phenyleneethynylene based polymers and oligomers. Soft Matter.8:8547-8558. Yeaman MR, Yount NY. 2003. Mechanisms of Antimicrobial Peptide Action and Resistence. Pharmacol. Rev.55:27-55. Miyata T, et al. 1989. Antimicrobial peptides, isolated from horseshoe cram hemocytes, tachyplesin II, and polyphemusin I and II: chemical structures and biological activity. J. Biochem.106:663-668. Powers JS, Martin MM, Goosney DL, Hancock REW. 2006. The antimicrobial peptide polyphemusin localizes to the cytoplasm of Escherichia coli following treatment. Antimicrob Agents Chemother. 4:1522-1524. Loose C, Jensen K, Rigoutsos I, Stephanopoulos G. 2006. A linguistic model for the rational design of antimicrobial peptides. Nature. 443: 867-869. Hancock REW and Sahl HG. 2006. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nature.24:1551-1557. Fjell CD, Hiss JA, Hancock REW, Schneider G. 2012. Designing antimicrobial peptides: form follows functions. Nature. 11:37-51.

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