Molnupiravir; molecular and functional descriptors of mitochondrial safety.

Molnupiravir is an orally active nucleoside analog antiviral drug that recently was approved by the U.S. FDA for emergency treatment of adult patients infected with the SARS-CoV-2 (COVID-19) virus and at risk for severe progression. The active form of the drug, N-hydroxycytidine (NHC) triphosphate competes for incorporation by RNA-dependent RNA-polymerase (RdRp) into the replicating viral genome resulting in mutations and arrest of the replicating virus. Historically, some nucleoside analog antiviral drugs have been found to lack specificity for the virus and also inhibit replication and/or expression of the mitochondrial genome. The objective of the present study was to test whether molnupiravir and/or NHC also target mitochondrial DNA polymerase gamma (PolG) or RNA polymerase (POLRMT) activity to inhibit the replication and/or expression of the mitochondrial genome leading to impaired mitochondrial function. Human-derived HepG2 cells were exposed for 48 h in culture to increasing concentrations of either molnupiravir or NHC after which cytotoxicity, mtDNA copy number and mitochondrial gene expression were determined. The phenotypic endpoint, mitochondrial respiration, was measured with the Seahorse® XF96 Extracellular Flux Analyzer. Both molnupiravir and NHC were cytotoxic at concentrations of ≥10 µM. However, at non-cytotoxic concentrations, neither significantly altered mitochondrial gene dose or transcription, or mitochondrial respiration. From this we conclude that mitochondrial toxicity is not a primary off target in the mechanism of cytotoxicity for either molnupiravir or its active metabolite NHC in the HepG2 cell line.

Remdesivir;molecular and functional measures of mitochondrial safety

Remdesivir is one of a few antiviral drugs approved for treating severe cases of coronavirus 2 (SARS-CoV-2) infection in hospitalized patients. The prodrug is a nucleoside analog that interferes with viral replication by inhibiting viral RNA-dependent RNA polymerase. The drug has also been shown to be a weak inhibitor of human mitochondrial RNA polymerase, leaving open the possibility of mitochondrial off-targets and toxicity. The investigation was designed to explore whether remdesivir causes mitochondrial toxicity, using both genomic and functional parameters in the assessment. Human-derived HepG2 liver cells were exposed for up to 48 h in culture to increasing concentrations of remdesivir. At sub-cytotoxic concentrations (<1 μM), the drug failed to alter either the number of copies or the expression of the mitochondrial genome. mtDNA copy number was unaffected as was the relative rates of expression of mtDNA-encoded and nuclear encoded subunits of complexes I and IV of the mitochondrial respiratory chain. Consistent with this is the observation that remdesivir was without effect on mitochondrial respiration, including basal respiration, proton leak, maximum uncoupled respiration, spare respiratory capacity or coupling efficiency. We conclude that although remdesivir has weak inhibitory activity towards mitochondrial RNA polymerase, mitochondria are not primary off-targets for the mechanism of cytotoxicity of the drug.