Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase

Abstract

ABSTRACTRibonucleotide analog inhibitors of the RNA-dependent RNA polymerase of hepatitis C virus (HCV) represent one of the most exciting recent developments in HCV antiviral therapy. Although it is well established that these molecules cause chain termination by competing at the triphosphate level with natural nucleotides for incorporation into elongating RNA, strategies to rationally optimize antiviral potency based on enzyme kinetics remain elusive. In this study, we used the isolated HCV polymerase elongation complex to determine the pre-steady-state kinetics of incorporation of 2′F-2′C-Me-UTP, the active metabolite of the anti-HCV drug sofosbuvir. 2′F-2′C-Me-UTP was efficiently incorporated by HCV polymerase with apparentKd(equilibrium constant) andkpol(rate of nucleotide incorporation at saturating nucleotide concentration) values of 113 ± 28 μM and 0.67 ± 0.05 s−1, respectively, giving an overall substrate efficiency (kpol/Kd) of 0.0059 ± 0.0015 μM−1s−1. We also measured the substrate efficiency of other UTP analogs and found that substitutions at the 2′ position on the ribose can greatly affect their level of incorporation, with a rank order of OH > F > NH2> F-C-Me > C-Me > N3> ara. However, the efficiency of chain termination following the incorporation of UMP analogs followed a different order, with only 2′F-2′C-Me-, 2′C-Me-, and 2′ara-UTP causing complete and immediate chain termination. The chain termination profile of the 2′-modified nucleotides explains the apparent lack of correlation observed across all molecules between substrate efficiency at the single-nucleotide level and their overall inhibition potency. To our knowledge, these results provide the first attempt to use pre-steady-state kinetics to uncover the mechanism of action of 2′-modified NTP analogs against HCV polymerase.