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 (<b>1</b>) as a potent antiviral agent against SARS-CoV-2 in Vero cells, with an EC₅₀ value of 1.71 μM and a CC₅₀ value exceeding 100 μM. Its triphosphate metabolite, compound <b>8</b>, inhibited the RNA-dependent RNA polymerase activity of the SARS-CoV-2 Nsp12-Nsp7-Nsp8₂ complex, terminating nascent RNA synthesis through misincorporation. Additionally, compound <b>8</b> 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 <b>1</b>. 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 <b>1</b> also exhibited broad-spectrum activity against various coronaviruses and other RNA viruses. These findings highlight that compound <b>1</b> is a promising orally available antiviral candidate.