HCV-NS3 inhibitors: Determination of their kinetic parameters and mechanism

Existing HCV protease inhibitors fall into two categories: reversible and non-covalent, such as BILN-2061, and covalent and reversible, exemplified by SCH-503034 and VX-950. In this work, the characterization of the kinetics of these three inhibitors is presented. SCH-503034 and VX-950 initially bind to the genotype 1b HCV NS3/4A protease to form a low affinity complex, with Ki values of 5 and 5.8 μM respectively. The ability of those two compounds to form a second covalent complex (EI⁎) results in a potency increase, with overall Ki⁎ values of 20 and 45 nM, respectively. The increase in potency can be explained by their slow dissociation rate, forming complexes with half-lives of 2 h (VX-950) and 5 h (SCH-503034). Although BILN-2061 has been described as a fast reversible, non-covalent inhibitor, our results show a slow binding two-step mechanism. Contrary to SCH-503034 and VX-950, BILN-2061 can form a high affinity first complex with a Ki value of 3.9 nM, and an overall Ki⁎ of 0.14 nM. The half-life of the BILN-2061 EI⁎ complex is shorter (t1/2 ∼ 0.7 h) than that of the other two compounds. The potency of these compounds is genotype dependent, and a kinetic analysis using NS3/4A from genotype 3a indicates that the loss of potency of SCH-503034 and VX-950 relative to genotype 1 is mainly due to the slow on-rate and faster off-rate for the formation of the EI⁎ complex. In the case of BILN-2061, a better fit is obtained using a one-step model, indicating that the loss of potency is due to an increase in the off-rate of the EI complex.