Hepatitis C NS3 protease inhibition by peptidyl-α-ketoamide inhibitors: kinetic mechanism and structure

A series of novel peptidyl-α-ketoamide compounds were evaluated as inhibitors of the ΔNS3-NS4A serine protease from the hepatitis C virus. These peptidyl-α-ketoamide inhibitors with Ki values ranging from 0.17 nM to 5.6 μM exhibited slow-binding inhibition. Kinetic studies established one-step kinetic mechanisms and dissociation rate constants in the 3–7 × 10−5 s−1 range for these compounds. The association rate constants, which ranged from 10 to 263,000 M−1 s−1, were responsible for the greater than four order of magnitude overall binding affinity range exhibited by this series. An X-ray crystal structure of a protease–inhibitor complex revealed an unusual interaction between the oxyanion of the adduct and the protein as well as a significant movement in the S1′ region of the protein loop comprising residues 35–42. These results are quite different from peptidyl-α-ketoacid inhibition of HCV protease, which reportedly undergoes no notable conformational changes and proceeds with a two-step slow-binding kinetic mechanism.