No abstract

The hepatitis C virus NS3 protein contains a serine protease domain with a chymotrypsin-like fold, which is a target for development of therapeutics. We report the crystal structures of this domain complexed with NS4A cofactor and with two potent, reversible covalent inhibitors spanning the P1-P4 residues. Both inhibitors bind in an extended backbone conformation, forming an antiparallel ␤-sheet with one enzyme ␤-strand. The P1 residue contributes most to the binding energy, whereas P2-P4 side chains are partially solvent exposed. The structures do not show notable rearrangements of the active site upon inhibitor binding. These results are significant for the development of antivirals. Hepatitis C virus (HCV)1 infection is a major health problem that leads to cirrhosis and hepatocellular carcinoma in a substantial number of infected individuals estimated at 100 -200 million worldwide. Immunotherapy or other effective treatments for HCV infection are not yet available, and administration of interferon in combination with ribavirin has several limitations because of toxicity (1). One of the best characterized targets for HCV therapy is the serine protease of NS3 protein. The NS3 protease domain constitutes the N terminus of the NS3 protein, which, when associated to the NS4A polypeptide, gets activated and therefore is responsible for maturation of the viral polyprotein (2).The structure determination of the HCV NS3 protease complexed with a truncated NS4A cofactor (residues 21-34) revealed a shallow, nonpolar P1 specificity pocket. Because of the unusual substrate specificity of this enzyme it has been inferred that the design of highly selective inhibitors that could bind to the NS3 protease would be unlikely (3, 4). We have found that capped tri-peptide ␣-ketoacids, incorporating difluoro aminobutyric acid in the P1 position, are potent, slow binding inhibitors of this enzyme (5). Their mechanism of inhibition is biphasic. The first kinetic phase involves the rapid formation of a noncovalent collision complex with association rate constants Ͼ0.2 s Ϫ1 , and the second kinetic phase consists of a slow isomerization with rate constants between 5 ϫ 10 Ϫ3 and 7.5 ϫ 10 Ϫ3 s Ϫ1 . This results in the formation of a very tight complex with dissociation rate constants between 1.2 ϫ 10 Ϫ5 and 1.8 ϫ 10Ϫ5 s Ϫ1 and with half-lives of 11-16 h. The overall K i values are between 27 and 67 nM (5).The inhibitors described here span the P1-P4 residues and contain an activated carbonyl in an ␣-ketoacid moiety as the active-site serine trap. To investigate the binding mode of these compounds, an hexagonal crystal form of the NS3 protease domain (J strain) complexed with the truncated NS4A cofactor, amenable to soaking experiments, was obtained. The crystal structures of the noninhibited NS3/4A complex (2.4 Å) and with two inhibitors (Fig. 1A), inhibitor I,were solved. EXPERIMENTAL PROCEDURESProtein Expression and Purification-A DNA fragment encoding the serine protease domain of NS3J (amino acids 1-187) was obtained by polymerase c...

The replication of the hepatitis C virus (HCV), an important human pathogen, crucially depends on the proteolytic maturation of a large viral polyprotein precursor. The viral nonstructural protein 3 (NS3) harbors a serine protease domain that plays a pivotal role in this process, being responsible for four out of the five cleavage events that occur in the nonstructural region of the HCV polyprotein. We here show that hexapeptide, tetrapeptide, and tripeptide alpha-ketoacids are potent, slow binding inhibitors of this enzyme. Their mechanism of inhibition involves the rapid formation of a noncovalent collision complex in a diffusion-limited, electrostatically driven association reaction followed by a slow isomerization step resulting in a very tight complex. pH dependence experiments point to the protonated catalytic His 57 as an important determinant for formation of the collision complex. K(i) values of the collision complexes vary between 3 nM and 18.5 microM and largely depend on contacts made by the peptide moiety of the inhibitors. Site-directed mutagenesis indicates that Lys 136 selectively participates in stabilization of the tight complex but not of the collision complex. A significant solvent isotope effect on the isomerization rate constant is suggestive of a chemical step being rate limiting for tight complex formation. The potency of these compounds is dominated by their slow dissociation rate constants, leading to complex half-lives of 11-48 h and overall K(i) values between 10 pM and 67 nM. The rate constants describing the formation and the dissociation of the tight complex are relatively independent of the peptide moiety and appear to predominantly reflect the intrinsic chemical reactivity of the ketoacid function.

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral RNA. We recently disclosed dihydroxypyrimidine carboxylates 2 as novel, reversible inhibitors of the HCV NS5B polymerase. This series was further developed into 5,6-dihydroxy-2-(2-thienyl)pyrimidine-4-carboxylic acids such as 34 (EC50 9.3 microM), which now show activity in the cell-based HCV replication assay. The structure-activity relationship of these inhibitors is discussed in the context of their physicochemical properties and of the polymerase crystal structure. We also report the results of mutagenesis experiments which support the proposed binding model, which involves pyrophosphate-like chelation of the active site Mg ions.

No abstract