A Novel Chemotype of Orally Active SARS-CoV-2 NSP14 Methyltransferase Inhibitors

The periodic emergence of human coronaviruses (hCoVs) necessitates direct-acting antivirals targeting underexploited viral proteins with broad-spectrum potential. Building on our discovery of TDI-015051, a first-in-class SARS-CoV-2 mRNA cap guanine-N7 methyltransferase (MTase) NSP14 inhibitor whose translational development was limited by metabolic liabilities, we conducted medicinal chemistry optimization of a structurally distinct high-throughput screening hit. We arrived at MAVDA-K-256, a small-molecule inhibitor with improved pharmacological properties compared to TDI-015051. MAVDA-K-256 engaged NSP14 through the formation of a ternary complex with S-adenosyl-l-homocysteine, potently inhibiting its MTase activity and SARS-CoV-2 replication (EC50 = 24.6 nM). MAVDA-K-256 demonstrated broad-spectrum antiviral activity against diverse hCoVs without targeting the host mRNA cap MTase. Its favorable metabolic stability and pharmacokinetic properties enabled twice-daily oral dosing in a murine SARS-CoV-2 infection model, resulting in a five-log reduction in viral lung burden. These findings validate NSP14 inhibition as a promising antiviral strategy and support further preclinical development of MAVDA-K-256.