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

Abstract

Ribonucleotide 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 apparent K d (equilibrium constant) and k pol (rate of nucleotide incorporation at saturating nucleotide concentration) values of 113 ؎ 28 M and 0.67 ؎ 0.05 s ؊1 , respectively, giving an overall substrate efficiency (k pol /K d ) of 0.0059 ؎ 0.0015 M ؊1 s ؊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 > NH 2 > F-C-Me > C-Me > N 3 > 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. . The primary mode of transmission for HCV is via exposure to infected blood, including transfusions from infected donors, and through intravenous use of illicit drugs. Although a minority of all HCV infections will spontaneously resolve without any clinical outcome, an estimated 80% of cases will progress into chronic hepatitis, leading to a significant proportion of cirrhosis and cases of hepatocellular carcinoma (2). This makes HCV the leading cause of liver transplantation in the United States.Hepatitis C virus is a member of the Flaviviridae family (3). It contains a single, positive-strand RNA genome of about 9.5 kb. The viral genome encodes only one open reading frame translated to a polyprotein of approximately 3,000 amino acids. The NS5B protein is composed of 591 amino acids that are cleaved at the C-terminal end of the polyprotein. NS5B acts as the RNA-dependent RNA polymerase (RdRp), with critical functions in RNA replication and transcription. Similar to other known RdRps, NS5B contains six conserved motifs, designated A through F. The amino acids involved in the catalytic activity of NS5B are located within motif A (aspartate at position 220) and in the catalytic triad GDD at positions 318 to ...

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