Identification of 4'-Thiouridine as an Orally Available Antiviral Agent Targeting Both RdRp and NiRAN Functions of SARS-CoV-2 Nsp12.

Through screening of a nucleos(t)ide-focused chemical library, we identified 4'-thiouridine () as a potent antiviral agent against SARS-CoV-2 in Vero cells, with an EC value of 1.71 μM and a CC value exceeding 100 μM. Its triphosphate metabolite, compound , inhibited the RNA-dependent RNA polymerase activity of the SARS-CoV-2 Nsp12-Nsp7-Nsp8 complex, terminating nascent RNA synthesis through misincorporation. Additionally, compound suppressed the function of the NiRAN domain of Nsp12, effectively blocking both RNAylation and NMPylation of Nsp9. Pharmacokinetic analysis in mice showed excellent oral bioavailability of compound . Oral administration at 100 mg/kg/day, twice daily for 5 days, protected mice from lethal SARS-CoV-2 infection, resulting in 40% survival and near-complete recovery of body weight by day 14 postinfection. Compound also exhibited broad-spectrum activity against various coronaviruses and other RNA viruses. These findings highlight that compound is a promising orally available antiviral candidate.