Benserazide, the first allosteric inhibitor of Coxsackievirus B3 3C protease

Abstract Coxsackievirus B3 is the main cause of human viral myocarditis and cardiomyopathy. Virally encoded Coxsackievirus 3C protease (3C^pro) plays an essential role in viral proliferation. Here, benserazide was discovered as a novel inhibitor from a drug library screen targeting Coxsackievirus 3C^pro using a FRET-based enzyme assay. Benserazide, whose chemical structure has no electrophilic functional groups, was characterized as a non-competitive inhibitor by enzyme kinetic studies. A molecular docking study with benserazide and its analogs indicated that a novel putative allosteric binding site was involved. Specifically, a 2,3,4-trihydroxybenzyl moiety was determined to be a key pharmacophore for the enzyme's inhibitory activity. We suggest that the putative allosteric binding site may be a novel target for future therapeutic strategies.