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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, 2010Synthesis and Folding of Kv1.3 Ion-Channel Blocking PeptidesG. Dello Iacono 1 , T. Leedom 1 , L. Wood 1 , D. Tumelty 1 , A. Bhat 1 ,G. Woodnutt 1 , Curt Bradshaw 1 , P. Morton 2 , R. Numann 2 , J. Ozer 2 ,G. Anderson 2 , G. Weber 2 , M. Schmidt 2 , Z.-L. Mo 2 , K. Keys 2 , B. Koci 2 ,M. Williams 2 , and J. Desharnais 11 CovX; A Pfizer BTx Division, 9381 Judicial Drive, Suite 200, San Diego, CA, U.S.A.;2 Pfizer 700 Chesterfield Pkwy, Chesterfield, Saint Louis, MO, U.S.A.IntroductionT cell-mediated autoimmune diseases afflict millions of people worldwide and includedisease such as multiple sclerosis (MS), rheumatoid arthritis (RA), psoriasis and others.Disease modifying immunotherapies have improved the management of autoimmunediseases, but these therapies induce a variety of unwanted side effects. Consequently, thereremains an enormous unmet medical need for novel immunomodulators with differentmechanisms of action and/or adverse-effect profiles from existing drugs. Preclinical andclinical evidence suggests that antagonizing Kv1.3 channels is effective in alleviating MSand RA syndromes. An example of such antagonists are the naturally-occurring toxinpeptides produced by a variety of organisms, such as snakes, scorpions, spiders, bees, snailsand sea anemones. In most cases, these toxin peptides have evolved as potent antagonists orinhibitors of ion-channels by binding to the channel pore and physically blocking the ionconduction pathway. Unfortunately due to their length (>35 aa) and complexity (most havemultiple disulfide bridges), they are often overlooked at the research stage. Here wedescribe the synthesis and folding of a peptide found in the venom of the Asian scorpionOrthochirus scrobiculosus and reported to be a potent antagonist of the Kv1.3 ion-channel.We also briefly describe the conjugation of this peptide to the antibody CovX-2000 aimedto enhance its pharmacokinetic properties.ResultsSynthesis and Folding of OSK1 toxinThe peptide was assembled stepwise using Fmoc/tBu chemistry starting with Rink amidepolystyrene resin. The Fmoc group was removed by a 20% piperidine/DMF solution. Allthe residues were double coupled using HBTU/HOBt/NMM in 1:1:4 ratio. The peptide wascleaved from the resin and simultaneously deprotected using theTFA/DTT/Phenol/TIPS/Water, 85: 5: 5: 2.5: 2.5 cocktail for 2 hrs at room temperature. For the folding a0.1M water solution of the peptide was made and a mix of Glutathione reduced/oxidized(GSH/GSSG 2:1 ratio) was added to a final concentration of 0.0005 M of each. The pHadjusted to around 8.3 using NH 4 OH. After 12 hr, no further conversion to the oxidizedform was observed. The cyclized peptide was estimated to be 75% by LC/MS analysis.(Figure 1).NNH 2GVSSOOO OI N V K N K I S R Q N L K P N K D A G M R F G K N M N G K N H N T POOSSSSNNOFig.1. Folded peptide.Peptide Peptide+linker Peptide +IgG1 CovX-BodyFig. 2. The CovX-Body concept.108