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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, 2010Development of a Protease-Resistant Bicyclic Peptide TargetingHuman Plasma KallikreinElise Bernard, David Loakes, and Gregory WinterLaboratory of Molecular Biology, Medical Research Council, Hills Road,Cambridge, CB2 0QH, United KingdomIntroductionMany kallikreins are involved in carcinogenesis making them interesting potentialbiomarkers for cancer. Human plasma kallikrein is a serine protease involved in hereditaryangioedema but only subcutaneous treatment is commercially available at the moment. Wehave been creating constrained bicyclic peptides; by constraining the peptide and engagingthe target with two loops we aim to improve their binding affinities over linear peptides.The constrained loops are also more difficult to cleave with proteases; however to get thebicyclic peptides through the proteases of the gut we need to make further improvements.We focused our research on modifying the sequence of a bicyclic kallikrein inhibitor toobtain a good resistance to proteases.Results and DiscussionWe obtain bicyclic peptides by nucleophilic substitution of linear sequences around abenzenic core. The three Cys residues substitute to trisbromomethylbenzene [1,2] to formthe constrained peptide. Our lead peptide, PK15 or ACSDRFRNCPADEALCG, is aconsensus sequence obtained by cloning and targeting human plasma kallikrein [3]. Weselected it for its excellent binding affinity of 4 nM. When cyclised, both loops are sixamino acids long.OBrBrBr+ONHSHNHONH 4 HCO 3 buffer / Acetonitrile(70/30)ONHSSNHSONHFig. 1. Synthesis of bicyclic peptides.HAlaHNCH 2CHSCH 2OSer AspArgPheH 2 H 2C S C HCCH 2SGlyHOCH 2CHNHOLoop 1Fig. 2. Bicyclic PK15.ArgAsnNHProLoop 2AlaAspLeu AlaGluOWe focused our research on the modification ofloop 1 of bicyclic PK15. This loop was shown to have agreater importance than the second loop as a truncatedPK15 containing only the first loop is 20 times morepotent than a truncated PK15 containing the secondloop. Loop 1 was highly conserved in clones selectedwith human plasma kallikrein [3].We designed two classes of peptidomimetics tofind some better protease resistant structures that couldresist a journey through the digestive system. Alterationof the side chain was investigated with peptidescontaining D-Phe, and where the Arg residues werereplaced by homoarginine known for introducing aresistance to trypsin [4]. Alteration of the backbone wasinvestigated with peptides containing NMe-Arg residues[5] and also with peptides having a reduced bondbetween Phe6 and Arg7 [6]. This modification wasobtained by reacting Fmoc-phenylalaninal with the freeamine of the peptide supported on the resin and reducingthe resulting imine bond with NaBH 4 /AcOH overnight [7].254