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

Abstract

Serine proteases are the most studied class of proteolytic enzymes and a primary target for drug discovery. Despite the large number of inhibitors developed so far, very few make contact with the prime site of the enzyme, which constitutes an almost untapped opportunity for drug design. In the course of our studies on the serine protease NS3/4A of hepatitis C virus (HCV), we found that this enzyme is an excellent example of both the opportunities and the challenges of such design. We had previously reported on two classes of peptide inhibitors of the enzyme: (a) product inhibitors, which include the P 6 -P 1 region of the substrate and derive much of their binding energy from binding of their C-terminal carboxylate in the active site, and (b) decapeptide inhibitors, which span the S 6 -S 4 ′ subsites of the enzyme, whose P 2 ′-P 4 ′ tripeptide fragment crucially contributes to potency. Here we report on further work, which combined the key binding elements of the two series and led to the development of inhibitors binding exclusiVely to the prime site of NS3/4A. We prepared a small combinatorial library of tripeptides, capped with a variety of constrained and unconstrained diacids. The SAR was derived from multiple analogues of the initial micromolar lead. Binding of the inhibitor(s) to the enzyme was further characterized by circular dichroism, site-directed mutagenesis, a probe displacement assay, and NMR to unequivocally prove that, according to our design, the bound inhibitor(s) occupies (occupy) the S′ subsite and the active site of the protease. In addition, on the basis of the information collected, the tripeptide series was evolved toward reduced peptide character, reduced molecular weight, and higher potency. Beyond their interest as HCV antivirals, these compounds represent the first example of prime site inhibitors of a serine protease. We further suggest that the design of an inhibitor with an analogous binding mode may be possible for other serine proteases.Serine proteases are perhaps the most studied class of proteolytic enzymes and a primary target for drug discovery. Despite the large number of inhibitors developed so far, very few make contact with the prime site of the enzyme. 1 Given that the starting point for inhibitor design is generally the substrate, this is not surprising, since for this class of enzymes the key determinants of substrate specificity reside in the P-region (1, 2-5). Therefore, developing inhibitors of serine proteases, which specifically target the prime site, is an almost untapped opportunity for drug design. In the course of our studies on the serine protease NS3/4A of hepatitis C virus (HCV), we found that this enzyme is an excellent example of both the opportunities and the challenges of such design.The target of our work is a key virally encoded enzyme, whose inhibition holds much promise for an effective anti-HCV therapy. Infection by hepatitis C virus (HCV) is widely recognized today as a huge public health concern, with more than 170 million people infe...

Identification of Residues in the Dengue Virus Type 2 NS2B Cofactor That Are Critical for NS3 Protease Activation

“…The result of these conformational changes is a reorientation of the residues of the catalytic triad which is more favorable for proton shuttling during catalysis. A number of studies provided evidence that structural rearrangements leading to a fully activated protease are induced not only by binding of the cofactor but also by the substrate, as shown for the competitive inhibition of the NS3 protease by its cleavage products (2,3,24,25,38). Based on these findings, the hepatitis C virus enzyme has been described as an induced-fit protease (6,39).…”

Inhibiting viral proteases: Challenges and opportunities

“…Prior to our work on NS3/4A, 44,46 other authors had already shown that prime site binding could be an effective component of a serine protease inhibitor, 48,49 but the previously described inhibitors all established crucial contacts in the nonprime region of the enzyme. The NS3 inhibitors described above represent therefore the first example of purely prime-site inhibitors of a serine protease.…”

“…These included kinetic analysis of competitive behavior, site-directed mutagenesis, comparison with the SAR of the P-PЈ inhibitors, a probe displacement assay, CD, and NMR, albeit in the absence of the NS4A cofactor. 46 CD in particular showed that binding of compound 1 to NS3/4A causes a change in the near-uv spectrum of the protease, with a small but consistent increase of intensity between 252 and 292 nm, indicating that, like for the P-PЈ decapeptides, an induced-fit mechanism is associated with the inhibition process. Selective N-methylation was then used to assess the backbone hydrogen-bonding pattern.…”

“…Therefore we decided to select the proper linker from a small combinatorial library. 46 The generic structure of the library was very simple, as shown in Figure 2: the P 2 Ј-P 4 Ј tripeptide fragment was capped with a variety of constrained and unconstrained diacids. Out of 26 components, we selected the only compound with a cyclic diacid,with IC 50 ϭ 54 M (Figure 2).…”

The Role of an Amphiphilic Capping Group in Covalent and Non-Covalent Dipeptide Inhibitors of HCV NS3 Serine Protease

“…Most of the inhibitors so far developed have as the starting point the hexapeptide product inhibitors of the enzyme [9][10][11]. Among them, a macrocyclic peptidomimetic shows a marked antiviral effect in clinical trials after oral administration [12].…”