Identification and Evaluation of Non-Nucleosidic MTase Inhibitors against SARS-CoV-2 nsp14 with Lower-Micromolar Anti-Coronavirus Activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes respiratory infections ranging from mild to severe, posing significant public health risks. The emergence of new variants highlights the need for inhibitors targeting conserved nonstructural proteins like nsp14, a key N7-methyltransferase (MTase) critical for viral RNA capping, immune evasion, and replication. Here, we screened 131 compounds using a drug repurposing approach and identified five candidates that inhibit MTase activity. Bobcat339 showed significant inhibition (IC₅₀ = 21.6 μM) and binding affinity (ΔT_m = +3.9 °C). It also reduced the replication of HCoV-229E and SARS-CoV-2 in infected Huh7 cells (EC₅₀ = 29.8 and 28.4 μM, respectively). Molecular docking suggested Bobcat339 binds the SAM-binding pocket of nsp14 MTase. These results identify Bobcat339 as a promising lead for developing selective, non-nucleoside nsp14 inhibitors, supporting further structural optimization and preclinical evaluation.