Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. The rapid development of highly effective vaccines2,3 against SARS-CoV-2 has altered the trajectory of the pandemic, and antiviral therapeutics4 have further reduced the number of COVID-19 hospitalizations and deaths. Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses that encode various structural and non-structural proteins, including those critical for viral RNA replication and evasion from innate immunity5. Here we report the discovery and development of a first-in-class non-covalent small-molecule inhibitor of the viral guanine-N7 methyltransferase (MTase) NSP14. High-throughput screening identified RU-0415529, which inhibited SARS-CoV-2 NSP14 by forming a unique ternary S-adenosylhomocysteine (SAH)-bound complex. Hit-to-lead optimization of RU-0415529 resulted in TDI-015051 with a dissociation constant (Kd) of 61 pM and a half-maximal effective concentration (EC50) of 11 nM, inhibiting virus infection in a cell-based system. TDI-015051 also inhibited viral replication in primary small airway epithelial cells and in a transgenic mouse model of SARS CoV-2 infection with an efficacy comparable with the FDA-approved reversible covalent protease inhibitor nirmatrelvir6. The inhibition of viral cap methylases as an antiviral strategy is also adaptable to other pandemic viruses.
