ABSTRACT
Molnupiravir (MOV) has received FDA's Emergency Use Authorization for the treatment of COVID-19, which is caused by SARS-CoV-2 infection. MOV is a prodrug of the ribonucleoside analog, Nhydroxycytidine (NHC). Upon phosphorylation, NHC incorporates into nascent viral RNA during replication triggering “catastrophic” mutation of the viral genome. However, NHC can also enter the deoxy-ribonucleotide pool, become incorporated into DNA, and cause DNA mutations. In nonclinical safety assessments, MOV was positive (i.e., mutagenic) in the Ames assay but negative in regulatory in vitro and in vivo micronucleus assays. Multiple in vitro studies conducted in bacteriophages, bacteria, fungi, and mammalian cells have reported that NHC can induce DNA mutations, mainly A:T>G:C transitions. We used a recently developed error-corrected wholegenome sequencing technique for detecting mutations induced by MOV and NHC in cultures of E. coli, mouse L5178YTk+/-, and human TK6 cells. Treatment of bacterial and mammalian cultures (for 4 hours and 5 days, respectively) with either MOV or NHC increased mutation frequencies in a dose-dependent manner in all three models. The majority of induced mutations were A:T>G:C, consistent with the type of mutation caused by incorporation of dNHC opposite to dA in the first round of DNA replication and incorporation of dG opposite to dNHC in the subsequent round(s) of DNA replication. Trinucleotide mutational signatures in MOV/NHC-treated cells were similar in mouse and human cells and different from the background spontaneous mutational signatures in parental cell cultures. The specific mutational signature was evident in mammalian cells exposed to NHC concentrations comparable to those observed in the plasma of human subjects who received clinical doses of MOV. This data indicates more well-controlled rodent and clinical studies of MOV/NHC-induced mutagenicity should be done in the interest of public health safety.