Inhibiting viral proteases: Challenges and opportunities

Abstract

Inhibitor design against viral targets must take into account the peculiar characteristics of viral biology-in particular, the plasticity of their replicative machinery. This includes maturational cleavage of the polyprotein, which is mediated by virally encoded proteases. Designing against a movable target is particularly challenging, but at the same time it offers new opportunities. Here we describe our experience with the NS3/4A (NS: nonstructural) serine protease of human hepatitis C virus (HCV). By extensive use of combinatorial peptide libraries, various inhibitor types were generated, including product inhibitors, serine traps, P-P' inhibitors, and prime side inhibitors. The latter represent a first case for a serine protease. A key finding, derived from structural studies utilizing these inhibitors, was that NS3 is an induced-fit protease, requiring both the NS4A cofactor protein and the substrate to fully activate its catalytic machinery. In the absence of cofactor and/or substrate, NS3 exists in solution as a large conformational ensemble, which can be matched by a correspondingly large set of peptide inhibitors, each one stabilizing a given conformer. In the perspective of inhibiting viral proteases in general, we suggest that combinatorial ligand ensembles may be a powerful tool, to contrast the adaptive potential of the viral quasispecies.

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

“…After extensive dialysis, wild-type NS2B(H)-NS3pro exhibited complete autoproteolytic cleavage, which resulted in two protein products of 20 kDa and 10 kDa, whereas the S135A mutant was completely inactive in the self-cleavage assay. In Western blot analysis, only the 10-kDa protein reacted with anti-Xpress antibodies directed against the polyhistidine tag, which confirmed that this protein represents the N-terminal cleavage fragment (His) 6 …”

“…Expression of the mutant derivatives as inclusion bodies in E. coli and purification under denaturing conditions by immobilized metal chelate chromatography and gel filtration yielded homogeneous products, as determined by SDS-PAGE analysis. The 29.8-kDa (His) 6 -NS2B(H)-NS3pro molecule displayed anomalous migration in gel electrophoresis with a higher apparent molecular mass of approximately 37 kDa. Subsequent refolding was performed by stepwise dialysis, and correct cleavage at the NS2B/NS3 junction was confirmed for the wild-type protein by N-terminal amino acid sequencing of the 20-kDa cleavage product, which yielded the sequence AGVLW, identical to the first five residues of the NS3 protein.…”

“…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). However, analogies to the hepatitis C virus system should be treated with caution, since preliminary data obtained with the dengue virus and the GB virus enzymes are indicative of major structural differences in the activation process (10,50).…”

Serpin Mechanism of Hepatitis C Virus Nonstructural 3 (NS3) Protease Inhibition

“…The interaction between the NS3 protease and its substrates is characterized by a series of weak contacts that extend over 10 amino acid residues, requires a cysteine in the P 1 position, and relies on high charge densities (17). Thus, substrate recognition by the NS3 protease is more reminiscent of the protein-protein interactions found in an enzyme-protein inhibitor pair than of a "conventional" enzyme-substrate complex (12,17).…”

“…The interaction between the NS3 protease and its substrates is characterized by a series of weak contacts that extend over 10 amino acid residues, requires a cysteine in the P 1 position, and relies on high charge densities (17). Thus, substrate recognition by the NS3 protease is more reminiscent of the protein-protein interactions found in an enzyme-protein inhibitor pair than of a "conventional" enzyme-substrate complex (12,17).In this article, we hypothesized that the bioengineering of a naturally occurring serine protease inhibitor (serpin) scaffold could provide a new approach to developing high affinity, selective NS3 protease inhibitors (18,19). Serpins differ from…”