Effects of the 5'-Triphosphate Metabolites of Ribavirin, Sofosbuvir, Vidarabine, and Molnupiravir on CTP Synthase Catalysis and Filament Formation: Implications for Repurposing Antiviral Agents against SARS-CoV-2.

ABSTRACT

Repurposing of antiviral drugs affords a rapid and effective strategy to develop therapies to counter pandemics such as COVID-19. SARS-CoV-2 replication is closely linked to the metabolism of cytosine-containing nucleotides, especially cytidine-5'-triphosphate (CTP), such that the integrity of the viral genome is highly sensitive to intracellular CTP levels. CTP synthase (CTPS) catalyzes the rate-limiting step for the de novo biosynthesis of CTP. Hence, it is of interest to know the effects of the 5'-triphosphate (TP) metabolites of repurposed antiviral agents on CTPS activity. Using E. coli CTPS as a model enzyme, we show that ribavirin-5'-TP is a weak allosteric activator of CTPS, while sofosbuvir-5'-TP and adenine-arabinofuranoside-5'-TP are both substrates. ß-d-N4 -Hydroxycytidine-5'-TP is a weak competitive inhibitor relative to CTP, but induces filament formation by CTPS. Alternatively, sofosbuvir-5'-TP prevented CTP-induced filament formation. These results reveal the underlying potential for repurposed antivirals to affect the activity of a critical pyrimidine nucleotide biosynthetic enzyme.