Mechanism of Inhibition for BMS-791325, a Novel Non-nucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase

HCV infection is an urgent global health problem that has triggered a drive to discover therapies that specifically target the virus. BMS-791325 is a novel direct antiviral agent specifically targeting HCV NS5B, an RNA-dependent RNA polymerase. Robust viral clearance of HCV was observed in infected patients treated with BMS-791325 in combination with other anti-HCV agents in Phase 2 clinical studies. Biochemical and biophysical studies revealed that BMS-791325 is a time-dependent, noncompetitive inhibitor of the polymerase. Binding studies with NS5B genetic variants (WT, L30S, and P495L) exposed a two-step, slow binding mechanism, but details of the binding mechanism differed for each of the polymerase variants. For the clinically relevant resistance variant (P495L), the rate of initial complex formation and dissociation is similar to WT, but the kinetics of the second step is significantly faster, showing that this variant impacts the final tight complex. The resulting shortened residence time translates into the observed decrease in inhibitor potency. The L30S variant has a significantly different profile. The rate of initial complex formation and dissociation is 7-10 times faster for the L30S variant compared with WT; however, the forward and reverse rates to form the final complex are not significantly different. The impact of the L30S variant on the inhibition profile and binding kinetics of BMS-791325 provides experimental evidence for the dynamic interaction of fingers and thumb domains in an environment that supports the formation of active replication complexes and the initiation of RNA synthesis.