Biochemical Characterization of the Active Anti-Hepatitis C Virus Metabolites of 2,6-Diaminopurine Ribonucleoside Prodrug Compared to Sofosbuvir and BMS-986094

Abstract

bRibonucleoside analog inhibitors (rNAI) target the hepatitis C virus (HCV) RNA-dependent RNA polymerase nonstructural protein 5B (NS5B) and cause RNA chain termination. Here, we expand our studies on ␤-D-2=-C-methyl-2,6-diaminopurine-ribonucleotide (DAPN) phosphoramidate prodrug 1 (PD1) as a novel investigational inhibitor of HCV. DAPN-PD1 is metabolized intracellularly into two distinct bioactive nucleoside triphosphate (TP) analogs. The first metabolite, 2=-C-methyl-GTP, is a wellcharacterized inhibitor of NS5B polymerase, whereas the second metabolite, 2=-C-methyl-DAPN-TP, behaves as an adenosine base analog. In vitro assays suggest that both metabolites are inhibitors of NS5B-mediated RNA polymerization. Additional factors, such as rNAI-TP incorporation efficiencies, intracellular rNAI-TP levels, and competition with natural ribonucleotides, were examined in order to further characterize the potential role of each nucleotide metabolite in vivo. Finally, we found that although both 2=-C-methyl-GTP and 2=-C-methyl-DAPN-TP were weak substrates for human mitochondrial RNA (mtRNA) polymerase (POLRMT) in vitro, DAPN-PD1 did not cause off-target inhibition of mtRNA transcription in Huh-7 cells. In contrast, administration of BMS-986094, which also generates 2=-C-methyl-GTP and previously has been associated with toxicity in humans, caused detectable inhibition of mtRNA transcription. Metabolism of BMS-986094 in Huh-7 cells leads to 87-fold higher levels of intracellular 2=-C-methyl-GTP than DAPN-PD1. Collectively, our data characterize DAPN-PD1 as a novel and potent antiviral agent that combines the delivery of two active metabolites. Chronic infection with hepatitis C virus (HCV) represents a significant disease burden on global health. It is estimated that 3% of the human population carries a chronic HCV infection, which translates to roughly 170 million infections worldwide and 3.2 million infections in the United States (1). Although up to 25% of acutely infected individuals clear the virus spontaneously, chronic HCV infection develops in the remaining 75% of those infected and can lead to the development of liver cirrhosis and hepatocellular carcinoma (2-4).HCV contains a 9.6-kb positive-strand RNA genome that codes for three structural and seven nonstructural proteins. The nonstructural protein 5B (NS5B) is the viral RNA-dependent RNA polymerase and is responsible for genome replication. Because of the high mutation rate of this enzyme, as well as the high virus replication rate, HCV propagation results in the generation of thousands of viral quasispecies (5, 6). Therefore, drug combination therapy is required in order to overcome development of resistance and to achieve successful viral clearance. HCV treatment comprised of ribavirin and pegylated interferon alpha, with or without protease inhibitors, can achieve sustained virologic responses in 40 to 80% of treated patients (7-9). However, curative outcomes historically have been suboptimal due to host polymorphisms, viral genotypic variability, and onset ...

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

“…Consistent with these observations, Arnold et al reported that increased POLRMT incorporation of NAI-TPs, as well as high intracellular NAI-TP levels, correlated positively with reductions in mitochondrial RNA levels and cytotoxicity (14). Similarly, we recently showed that treatment of Huh-7 cells with BMS-986094, which generates high levels of 2=-C-methyl-GTP intracellularly, inhibited mitochondrial RNA transcription (17). Interestingly, treatment with a 2,6-diaminopurine nucleotide (DAPN) prodrug, an investigational anti-HCV agent that also generates 2=-C-methyl-GTP (18), did not have similar deleterious effects.…”

“…Interestingly, treatment with a 2,6-diaminopurine nucleotide (DAPN) prodrug, an investigational anti-HCV agent that also generates 2=-C-methyl-GTP (18), did not have similar deleterious effects. Lower levels of intracellular NAI-TP accumulation were proposed to account for the distinct cytotoxicity profiles of these compounds (17).…”

“…1). As described previously (14,17), 125 nM purified POLRMT enzyme was incubated with 500 nM 5=-radiolabeled RNA/DNA primer/ template that allowed the incorporation of A, C, G, or U nucleotide analogs at position ϩ1. Each nucleoside triphosphate (NTP) analog (100 M) was incubated for 2 h at 30°C with the POLRMT-RNA/DNA complex containing the appropriate DNA template.…”

“…2C), N1-methyl-GTP was selected for further analysis with regard to incorporation by viral nonstructural protein 5B (NS5B) RNA polymerase. NS5B incorporation assays were performed as described previously (17). NS5B-RNA/ RNA complexes were incubated with increasing concentrations of GTP or N1-methyl-GTP.…”

Characterization of β- d - N 4 -Hydroxycytidine as a Novel Inhibitor of Chikungunya Virus

“…We next evaluated the intracellular metabolism of NHC. We incubated a 10 M or 50 M concentration of the NHC parent nucleoside in Huh-7 cells essentially as described previously (20). Briefly, Huh-7 cells were seeded at 1 ϫ 10 6 cells per well in 12-well plates and incubated with NHC for 4 h. Cells were subsequently washed and analyzed using liquid chromatography-tandem mass spectroscopy (LC-MS/MS).…”