Optimization of the P '-region of peptide inhibitors of hepatitis C virus NS3/4A protease

Infection by Hepatitis C Virus (HCV) leads to a slowly progressing disease that over two decades can lead to liver cirrhosis or liver cancer. Currentl y, one of the most promising approaches to anti-HCV therapy is the developm ent of inhibitors of the NS3/4A protease, which is essential for maturation of the viral polyprotein. Several substrate-derived inhibitors of NS3/4A h ave been described, all taking advantage of binding to the S subsite of the enzyme. Inspection of the S' subsite of NS3/4A shows binding pockets which might be exploited for inhibitor binding, but due to the fact that ground- state binding to the S' subsite is not used by the substrate, this does not represent a suitable starting point. We have now optimized S'-binding in t he context of noncleavable decapeptides spanning P6-P4'. Binding was sequen tially increased by introduction of the previously optimized P-region [Inga llinella et al. (1998) Biochemistry 37, 8906-8914], change of the P4' resid ue, and combinatorial optimization of positions P2'-P3'. The overall proces s led to an increase in binding of more than 3 orders of magnitude, with th e best decapeptide showing IC50 < 200 pM. The binding mode of the decapepti des described in the present work shares features with the binding mode of the natural substrates, together with novel interactions within the S' subs ite. Therefore, these peptides may represent an entry point for a novel cla ss of NS3 inhibitors.