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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Simplified Θ-Defensins: Search for New AntiviralsPiotr Ruchala 1 , Sylvia Cho 3 , Amy L. Cole 4 , Chun-Ling Jung 1 ,Hai T. Luong 1 , Ewa D. Micewicz 2 , Alan J. Waring 1 ,Alexander M. Cole 4 , Betsy C. Herold 3 , and Robert I. Lehrer 11 Department of Medicine; 2 Radiation Oncology, David Geffen School of Medicine,University of California at Los Angeles, Los Angeles, CA, 90095, U.S.A.; 3 Department ofPediatrics and Microbiology-Immunology, Albert Einstein College of Medicine of YeshivaUniversity, New York, NY, 10461, U.S.A.; 4 Department of Molecular Biology andMicrobiology, Biomolecular Science Center, University of Central Florida,Orlando, FL, 32816, U.S.A.IntroductionThree subfamilies of defensins exist in vertebrates, and are known as α-, β-, and θ-defensins [1]. They are small, amphipathic and cationic peptides with largely β-sheetstructures which are stabilized by 3 intramolecular disulfide bonds. Human α- and β-defensins have broad antibacterial and antifungal activity as well as prominent antiviralactivity against HIV-1, HSV-2 and influenza A [1]. The third subfamily, θ-defensins, is notexpressed in humans. It was initially isolated from the leukocytes and bone marrow ofnonhuman primates, specifically Old World monkeys. Subsequent numerous studiesestablished that θ-defensins are effective primarily against viruses acting as potent entryinhibitors for HIV-1, HSV-2 and influenza A [2,3]. Despite small size (18 Xaa), θ-defensins are difficult to synthesize. Chemically, they belong to cysteine-rich peptides andin addition contain a circular backbone. Such structure requires 2 post-synthetic steps:oxidation (disulfide bonds formation) and cyclization (circularization via amide bond)resulting in a low final yield of desired product. Development of an expression systemcapable of delivering recombinant θ-defensins was also unsuccessful to date hamperinggreatly their development as potential antiviral drugs. Therefore our current studies focusedon modification of θ-defensins with the general goal to simplify their structure and preserveor possibly enhance their antiviral properties.Results and DiscussionWe hypothesized that the anti-viral properties of retrocyclins might rely largely onstructural features located in the central β-sheet region, which is defined by three disulfidebridges. Accordingly, we designed and synthesized two mini-libraries of smaller analoguescalled Hapivirins (HpVs) and Diprovirins (DpVs). The HpVs and DpVs consist of 13residues and do not possess a cyclic backbone. In the case of DpVs, the β-turn and hairpinstructure resulted from an introduction of (D)Pro-(L)Pro dipeptide moiety. HPVs and DpVsare easier to produce, and in the case of DpVs, may potentially be made and oxidizedentirely on the resin, reducing post-synthetic work considerably, giving high final yields ofdesired compound(s).HpVs and DpVs (Figure 1, 70variants) were screened for activityagainst HIV-1 and HSV-2 usingTZM-bl assay and plaque assayrespectively (Table 1). The mostactive compound, DpV16, possessesantiviral activity comparable toparental θ-defensins (RC1 andRC101). Hydrophobicity in positionsX 2 and X 3 is important forantiviral activity since onlyanalogues carrying Ile and Leu in X 2and Ach, Tle, Cpg, Chg and Tyr inFig. 1. Structure of Hapivirins (HpVs) andDiprovirins (DpVs). X 1 , X 2 , X 3 , X 4 , X 5 - variableresidues.X 3 were active. Only one additionalpermissive substitution utilizing Argresidues was found (DpV1622).Oxidation via disulfide bond558