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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, 2010Design, Synthesis and Stability Studies of Potent Inhibitors ofPro-Protein Convertases (PCs)Anna Kwiatkowska 1 , Nicholas Chevalier 2 , Roxane Desjardins 2 ,Frédéric Couture 2 , François D’Anjou 2 , Sophie Routhier 2 ,Christine Levesque 2 , Yves Dory 2 , Witold Neugebauer 2 ,and Robert Day 21 Faculty of Chemistry, University of Gdańsk, Sobieskiego 18, 80-952, Gdańsk, Poland;2 Institut de Pharmacologie de Sherbrooke, Faculté de médecine et des sciences de la santé3001, 12e Avenue Nord Sherbrooke, Qc, J1H 5N4, CanadaIntroductionPro-protein convertases (PCs) are a mammalian family of serine endoproteases responsiblefor post-translational processing of inactive precursors of many regulatory proteins. Sevenstructurally related PCs, known as furin, PACE4, PC1/3, PC2, PC4, PC5/6 and PC7 cleavevarious precursors at the motif (K/R)-(X) n -(K/R) [1]. Several important pathologies havebeen linked with PC-like activity such as cancer and viral and bacterial, infections. Wehave previously developed a relatively specific inhibitor of PACE4, PC5/6 and PC7(Figure 1) [2]. The introduction of multi-Leu residues in the P8-P5 positions made thepeptide more selective for these enzymes in comparison to Furin and PC2. This inhibitorhas potent effects on cell proliferation and tumor progression, especially in cell models ofprostate cancer [2]. However further stabilization of the structure is necessary for potentialin vivo use.Ac-Leu-Leu-Leu-Leu-Arg-Val-Lys-Arg-NH 2Fig. 1. Structure of multi-Leu inhibitor.ObjectivesThe aim of this work was to introduce a series of chemical modifications in the multi-Leupart of our potent PC inhibitor. Several approaches including the use of D-amino acids(D-Leu), unusual amino acids (Nle), and click chemistry have been used to improve thepharmacokinetic properties of the model peptide (Figure 2).Fig. 2. Modifications of multi-Leu inhibitor.We synthesized 14 new analogues of multi-Leu inhibitor: Ac-[D-Leu 1 ]LLLRVKR-NH 2 (I),Ac-L[D-Leu 2 ]LLRVKR-NH 2 (II), Ac-LL[D-Leu 3 ]LRVKR-NH 2 (III), Ac-LLL[D-Leu 4 ]RVKR-NH 2 (IV), Ac-[D-Leu 1 ,D-Leu 2 ,D-Leu 3 ,D-Leu 4 ]RVKR-NH 2 (V), Ac-[Nle 1 ]LLLRVKRNH 2 (VI), Ac-L[Nle 2 ]LLRVKR-NH 2 (VII), Ac-LL[Nle 3 ]LRVKR-NH 2 (VIII), Ac-LLL[Nle 4 ]RVKR-NH 2 (IX), Ac-[Nle 1 ,Nle 2 ,Nle 3 ,Nle 4 ]RVKR-NH 2 (X), Ac-L1,4[1,2,3(triazole)]LLLRVKR-NH 2 (XI), Ac-LL1,4[1,2,3(triazoil)]LLRVKR-NH 2 (XII), Ac-LLL1,4[1,2,3(triazoil)]LRVKR-NH 2 (XIII), Ac-L1,4[1,2,3(triazole)]LL1,4[1,2,3(triazole)]LRVKR-NH 2(XIV) and determined their inhibitory potency against PCs. Furthermore, we characterizedthe stability and bioavailability of selected compounds.Results and DiscussionPeptides were synthesized using standard Fmoc/tBu solid phase procedures. The analysis ofinhibition was done in duplicate and is presented as the mean of three or more independentexperiments (Figure 3). Quantitative data analysis was done using four-parameter logisticand real inhibition constants (K i ) were determined using the method and K m valuesdescribed previously [3].466