α-Ketoacids Are Potent Slow Binding Inhibitors of the Hepatitis C Virus NS3 Protease

Abstract

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.

Preclinical Profile of VX-950, a Potent, Selective, and Orally Bioavailable Inhibitor of Hepatitis C Virus NS3-4A Serine Protease

“…The inhibition constant describing the equilibrium between the covalent tightly bound complex and free enzyme plus inhibitor (K i *) has been determined to be 7 nM for VX-950 with the NS3 protease domain and KK4A peptide of the HCV H strain under steady-state conditions, which is comparable to that of BILN 2061 (10 nM) in our laboratory. A similar mechanism of inhibition has been described previously for other ␣-ketoacid or ␣-ketoamide inhibitors of HCV NS3-4A protease (3,27,33).…”

Characterization of the Hepatitis C Virus NS2/3 Processing Reaction by Using a Purified Precursor Protein

“…To this end, we used the H952A mutant that did not show detectable NS2/3 protease activity. The NS3 protease active-site concentration in our refolded NS2/3 (907-1206) ASK 4 H952A preparation was determined by titration with a tight-binding NS3 protease inhibitor (26) and was used to normalize protein concentrations. Next, the affinity for an NS3 protease NS4A cofactor peptide (34) and the kinetic parameters for the hydrolysis of two NS3 peptide substrates were determined for both the NS3 protease domain (residues 1026 to 1207 of the HCV polyprotein) and our truncated NS2/3 precursor (residues 907 to 1207).…”

In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

“…Protein stocks were stored at a concentration of 10 to 20 M in 25 mM HEPES (pH 7.4)-1 mM EDTA-20% glycerol-3 mM DTT-0.1% dodecyl-maltoside-1 M NaCl at Ϫ80°C after being shock frozen in liquid nitrogen. The enzyme concentration was determined by active-site titration experiments using a hexapeptide ␣-ketoacid (38).…”

“…Similarly to compound 1, compound 2 has an alpha carboxylic acid and is a well-characterized product inhibitor (38,39). In compound 3, the P1 carboxylic acid is replaced by an alpha ketoacid, resulting in a covalent inhibitor of the protease (38). In compound 4, the alpha carboxylic acid is replaced by phenethyl-amide (10).…”

“…These inhibitors were identified based on the observation that NS3-4Ap was competitively inhibited by the N-terminal cleavage products, which had affinities for the enzyme higher than those of the corresponding substrates (31,45). These inhibitors have been subsequently optimized by progressive trimming of the original hexapeptides to tetra-or tripeptides, in parallel with replacing the side chains with unnatural substituents that significantly increased affinity for the enzyme (19,29,30,38 . More recently, further structure-activity studies have yielded potent and selective inhibitors that begin to have properties suitable for a drug (48).…”