Role of Mitochondrial RNA Polymerase in the Toxicity of Nucleotide Inhibitors of Hepatitis C Virus

Abstract

Toxicity has emerged during the clinical development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clinically relevant HCV NI were characterized in biochemical and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4′-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2′-C-methyl guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concentrations, followed by general cytotoxicity. In contrast, NI containing multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2′-deoxynucleotide analogs, there is an association between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clinical testing.

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

“…In this study, we aimed to look more closely at correlates of cytotoxicity with BMS-986094 treatment in order to better understand the lack of cytotoxicity ob- served with DAPN phosphoramidate prodrugs. It was recently reported that human mitochondrial RNA polymerase (POLRMT) incorporates 2=-C-methyl-GTP as well as a number of other anti-HCV ribonucleoside analogs (32,33). Here, we show that 2=-Cmethyl-DAPN-TP is also a substrate for POLRMT (Fig.…”

“…Primary human hepatocytes were estimated to contain amounts of natural NTPs comparable to those of Huh-7 cells. Nucleotide levels measured by LC-MS/MS were converted to micromolar concentrations, estimating a cell volume of 2 pl/cell (33). b Intracellular 2=-C-methyl-DAPN-TP and 2=-C-methyl-GTP levels measured in primary human hepatocytes after treatment with 50 M DAPN-PD1 for 24 h. c Rate constant for incorporation was determined with the equation…”

“…Intracellular 2=-C-methyl-GTP levels generated from each prodrug were determined using LC-MS/ MS. Each value is the average from two separate replicates (Ϯ standard deviations). b Amount of nucleoside 5=-triphosphate metabolites was converted from picomoles per million cells to molarity using an estimated cellular volume of 2 pl per cell (33). c Rate constant for incorporation was determined with the equation…”

“…K eff, POLRMT was calculated based on reported kinetic properties of 2=-C-methyl-GTP incorporation by POLRMT enzyme (33). d Selectivity was determined as (K eff, POLRMT ) 2=-C-methyl-GTP from DAPN-PD1 / (K eff, POLRMT ) 2=-Cmethyl-GTP from BMS-986094 .…”

“…In agreement with this hypothesis, recent studies have shown that human mtRNA polymerase (POLRMT) can indeed incorporate ribonucleoside 5=-triphosphate analogs in vitro. This is in turn correlated with inhibition of mtRNA transcription and interference with mitochondrial function in cell culture (32,33). Thus, mechanisms associated with ribonucleotide analog toxicity are of significant interest as new rNAI are being developed for the treatment of infections with RNA viruses.…”