Structural Modification of Tranilast against Poxvirus Based on α, β-Unsaturated Acrylamide Covalent Warhead and Confirmation of the Targets and Mode of Action.

The recurrent mpox outbreak has raised widespread global concern, yet specific antiviral therapies remain unavailable. Based on the α, β-unsaturated acrylamide covalent warhead of tranilast (TRA), we designed and synthesized 24 new TRA derivatives and evaluated their anti-orthopoxvirus activity using a VTT-Fluc assay. Compound 8l exhibited the most potent antipoxvirus activity and moderate activity against monkeypox clade IIb, with EC50 values of 6.1 and 47.1 μM, respectively, significantly outperformed TRA. In a VTT-Fluc nude mouse model, oral administration of 8l at 90 mg/kg achieved an 88.2% viral inhibition rate. Mechanistic studies revealed 8l exerted dual-target inhibition by acting on viral A17L and mRNA methyltransferase, thereby blocking membrane fusion and intracellular biosynthesis. The acrylamide warhead in 8l forms covalent adducts with serine residues of both targets via Michael addition. This work provides a promising covalent lead and identifies potential targets for the development of broad-spectrum anti-orthopoxvirus agents.