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Peptide C-terminal thioesters are key intermediates in a variety of applications, most notably the recently developed native chemical ligation methods for the total chemical synthesis of proteins. So far they have been prepared only by the use of the least prevalent Boc/benzyl solid-phase method on ad hoc prepared resin supports. We describe here a novel method for the solid phase synthesis of thioesters by the most prevalent Fmoc/t-Bu method. The method is based on the use of a 3-carboxypropanesulfonamide safety-catch linker, which is fully stable to repetitive exposure to the basic conditions needed for Fmoc cleavage. Activation with diazomethane or iodoacetonitrile followed by displacement with a suitable thiol produces the thioester in good to excellent yields. The method is also compatible with Boc/benzyl chemistry. Moreover, all the necessary reagents are commercially available.

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.

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One of the most promising approaches to anti-hepatitis C virus drug discovery is the development of inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is essential for the maturation of the viral polyprotein, and processing requires complex formation between NS3 and its cofactor NS4A. Recently, we reported on the discovery of potent cleavage product-derived inhibitors [Ingallinella et al. (1998) Biochemistry 37, 8906-8914]. Here we study the interaction of these inhibitors with NS3 and the NS3/cofactor complex. Inhibitors bind NS3 according to an induced-fit mechanism. In the absence of cofactor different binding modes are apparent, while in the presence of cofactor all inhibitors show the same binding mode with a small rearrangement in the NS3 structure, as suggested by circular dichroism spectroscopy. These data are consistent with the hypothesis that NS4A complexation induces an NS3 structure that is already (but not entirely) preorganized for substrate binding not only for what concerns the S' site, as already suggested, but also for the S site. Inhibitor binding to the NS3/cofactor complex induces the stabilization of the enzyme structure as highlighted by limited proteolysis experiments. We envisage that this may occur through stabilization of the individual N-terminal and C-terminal domains where the cofactor and inhibitor, respectively, bind and subsequent tightening of the interdomain interaction in the ternary complex.

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Infection by Hepatitis C Virus (HCV) leads to a slowly progressing disease that over two decades can lead to liver cirrhosis or liver cancer. Currently, one of the most promising approaches to anti-HCV therapy is the development of inhibitors of the NS3/4A protease, which is essential for maturation of the viral polyprotein. Several substrate-derived inhibitors of NS3/4A have 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 the context of noncleavable decapeptides spanning P6-P4'. Binding was sequentially increased by introduction of the previously optimized P-region [Ingallinella et al. (1998) Biochemistry 37, 8906-8914], change of the P4' residue, and combinatorial optimization of positions P2'-P3'. The overall process led to an increase in binding of more than 3 orders of magnitude, with the best decapeptide showing IC(50) < 200 pM. The binding mode of the decapeptides described in the present work shares features with the binding mode of the natural substrates, together with novel interactions within the S' subsite. Therefore, these peptides may represent an entry point for a novel class of NS3 inhibitors.