alpha-ketoacids are potent slow binding inhibitors of the hepatitis C virus NS3 protease

The replication of the hepatitis C virus (HCV), an important human pathogen , crucially depends on the proteolytic maturation of a large viral polyprot ein precursor. The viral nonstructural protein 3 (NS3) harbors a serine pro tease domain that plays a pivotal role in this process, being responsible f or four out of the five cleavage events that occur in the nonstructural reg ion of the HCV polyprotein. We here show that hexapeptide, tetrapeptide, an d tripeptide alpha-ketoacids are potent, slow binding inhibitors of this en zyme. Their mechanism of inhibition involves the rapid formation of a nonco valent collision complex in a diffusion-limited, electrostatically driven a ssociation reaction followed by a slow isomerization step resulting in a ve ry tight complex. pH dependence experiments point to the protonated catalyt ic His 57 as an important determinant for formation of the collision comple x. K-i values of the collision complexes vary between 3 nM and 18.5 mu M an d largely depend on contacts made by the peptide moiety of the inhibitors. Site-directed mutagenesis indicates that Lys 136 selectively participates i n stabilization of the tight complex but not of the collision complex. A si gnificant solvent isotope effect on the isomerization rate constant is sugg estive 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* value s between 10 pM and 67 nM. The rate constants describing the formation and the dissociation of the tight complex are relatively independent of the pep tide moiety and appear to predominantly reflect the intrinsic chemical reac tivity of the ketoacid function.