Potent Peptide Inhibitors of Human Hepatitis C Virus NS3 Protease Are Obtained by Optimizing the Cleavage Products

Abstract

In the absence of a broadly effective cure for hepatitis caused by hepatitis C virus (HCV), much effort is currently devoted to the search for inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is required for the maturation of the viral polyprotein, cleaving it at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. In the course of our studies on the substrate specificity of NS3, we found that the products of cleavage corresponding to the P6-P1 region of the substrates act as competitive inhibitors of the enzyme, with IC50s ranging from 360 to 1 microM. A detailed study of product inhibition by the natural NS3 substrates is described in the preceding paper [Steinkühler, C., et al. (1997) Biochemistry 37, 8899-8905]. Here we report the results of a study of the structure-activity relationship of the NS3 product inhibitors, which suggest that the mode of binding of the P region-derived products is similar to the ground-state binding of the corresponding substrates, with additional binding energy provided by the C-terminal carboxylate. Optimal binding requires a dual anchor: an "acid anchor" at the N terminus and a "P1 anchor" at the C-terminal part of the molecule. We have then optimized the sequence of the product inhibitors by using single mutations and combinatorial peptide libraries based on the most potent natural product, Ac-Asp-Glu-Met-Glu-Glu-Cys-OH (Ki = 0.6 microM), derived from cleavage at the NS4A-NS4B junction. By sequentially optimizing positions P2, P4, P3, and P5, we obtained several nanomolar inhibitors of the enzyme. These compounds are useful both as a starting point for the development of peptidomimetic drugs and as structural probes for investigating the substrate binding site of NS3 by modeling, NMR, and crystallography.

Prime Site Binding Inhibitors of a Serine Protease: NS3/4A of Hepatitis C Virus

“…The most important perturbations were observed around Ser 42 (the region from Ser 37 to Thr 46 ) and around two of the members of the catalytic triad (Asp 81 and Ser 139 ). The shifts observed for these residues are similar to those previously measured (35), highlighting that both the old (2,15,35) and the new inhibitors occupy the active site of the enzyme. By contrast, no significant shift was observed in the previously defined S region of the enzyme (residues Ala 156 to Val 170 ), in striking contrast with the binding of inhibitors based on the P region of the substrate (35).…”

“…Overall, all of the above data were highly suggestive that, according to our design, the capped tripeptides bound to the S′ region of the enzyme in a manner very similar to the decapeptides from which they derived, with their N-terminal carboxylate positioned in the active site, as observed for the product inhibitors (2,15).…”

“…These shifts were used to map the interaction surface between the enzyme and the inhibitor, characterizing the region of the protein involved in compound recognition. This study was performed in the absence of the cofactor, since the complex between NS3 and Pep4AK peptide (2,3,15,23) is not stable enough in solution to allow characterization by NMR (21).…”

“…Accordingly, we (2,15) as well as others (16,17) were able to develop potent peptide inhibitors derived from the nonprime region of the substrate. By contrast, the prime region of the substrate, while being important for catalysis, contributes little to groundstate binding (3)(4)(5).…”

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism

“…However, an environment similar to that of the longchain subfamily could be partially created with the participation of the substrate. We observe, in fact, for the short-chain enzymes a tendency to have a hydrophobic bulky residue in position P2 (Tong et al, 1993;Ingallinella et al, 1998), whereas longchain enzymes tend to have either glycine or short side-chain residues such as Ala (Yasutake & Powers, 1981;Chang, 1986;Coombs et al, 1996). A bulky residue in position P2 together with the aliphatic methylene groups of R155 (NS3) and of the L231 side-chain (Sindbis and Semliki viruses) could contribute to shelter the aspartic acid side-chain, thereby favouring the formation of a catalytic triad machinery that more closely resembles that observed in long-chain enzymes.…”