Nucleotide Substrate Specificity of Anti-Hepatitis C Virus Nucleoside Analogs for Human Mitochondrial RNA Polymerase

Abstract

Nucleoside analog inhibitors (NAIs) are an important class of antiviral agents. Although highly effective, some NAIs with activity against hepatitis C virus (HCV) can cause toxicity, presumably due to off-target inhibition of host mitochondrial RNA polymerase (POLRMT). The in vitro nucleotide substrate specificity of POLRMT was studied in order to explore structure-activity relationships that can facilitate the identification of nontoxic NAIs. These findings have important implications for the development of all anti-RNA virus NAIs.

Keywords: antiviral agents; hepatitis C virus; toxicity.

FIG 1

Chemical structures of NAI-TPs. (A) Structures of NAI-TPs with modifications on the ribose moiety. Compounds are grouped according to the base moiety. (B) Structures of dNAI-TPs with anti-HIV, anti-HBV, or anti-HSV activity. (C) Structures of rNTP analogs with modifications on the base moiety.

FIG 2

NTP analog incorporation by POLRMT. (A) NTP analogs with modifications at the 2′ position of the ribose moiety were assessed for incorporation by POLRMT. Incorporation reactions were allowed to proceed for 2 h in the presence of 100 μM each substrate. Percentage incorporation of each NTP analog was normalized to that of the corresponding natural nucleotide substrate (ATP, CTP, GTP, or UTP). (B) POLRMT incorporation of 2′-deoxyribonucleoside analogs and NAI-TPs with anti-HBV, anti-HIV, or anti-HSV activity was assessed as described above. (C) POLRMT incorporation was assessed for rNTP analogs with modifications on the base moiety. Error bars represent standard deviations (SDs) for two or three separate experiments.

FIG 3

Incorporation profile for N1-methyl-GTP. (A) Increasing concentrations of GTP (0.7 μM to 180 μM) were incubated with a preformed NS5B-RNA/RNA complex, and nucleotide extension was measured over time. The extended 10-mer RNA product was visualized on a 20% denaturing acrylamide gel. Rates of incorporation at various nucleotide concentrations were plotted as described previously, in order to obtain an apparent dissociation constant (K_d,app) value of 4.1 ± 1.6 μM for GTP (17). (B) Increasing concentrations of N1-methyl-GTP (15.6 μM to 500 μM) were incubated with the preformed NS5B-RNA/RNA complex as described above. The K_d,app value for N1-methyl-GTP was estimated to be >100 μM.

FIG 4

Effect of N1-methyl-GTP incorporation on NS5B-mediated RNA extension. (A) Increasing concentrations of N1-methyl-GTP were incubated with the NS5B enzyme, 5′-radiolabeled GG primer, and a 20-mer RNA template in the presence of 10 μM ATP, CTP, and UTP. RNA synthesis was allowed to proceed for 2 h at 30°C. In the absence of N1-methyl-GTP (lane 0), a strong pausing site was observed at position 9, while small amounts of full-length 20-mer RNA product accumulated as a result of nucleotide misincorporation. Increasing N1-methyl-GTP concentrations were correlated with the appearance of a 10-mer band (site of G incorporation) and the full-length 20-mer product. (B) Amounts of 20-mer product accumulation were plotted as a function of the N1-methyl-GTP concentration (left). Data from parallel experiments with increasing concentrations of GTP were also plotted (right). (C) RNA synthesis was monitored as described above, with the addition of 1 μM GTP in the presence of increasing concentrations of N1-methyl-GTP (left) or the control inhibitor 2′-C-methyl-GTP (right). No inhibition of RNA synthesis was observed with up to 1,000 μM N1-methyl-GTP, while 2′-C-methyl-GTP inhibited RNA synthesis with an IC₅₀ of 3.3 ± 0.5 μM (average ± SD of two separate experiments).

FIG 5

Chemical synthesis of the N1-methyl-G phosphoramidate prodrug. Step a, p-toluenesulfonic acid and 2,2-dimethoxypropane in acetone, overnight at room temperature (yield, 93%); step b, NaH and MeI in dimethyl sulfoxide, overnight at room temperature (yield, 90%); step c, phosphorochloridate and N-methylimidazole in tetrahydrofuran-acetonitrile, 3 h at room temperature (yield, 91%); step d, 85% trifluoroacetic acid, 1 h at room temperature (yield, 86%).