Abstract

SARS-CoV-2 main protease (Mᵖʳᵒ) is one of the most extensively exploited drug targets for COVID-19. Structurally disparate compounds have been reported as Mᵖʳᵒ inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed Mᵖʳᵒ inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based FlipGFP assay.

Collectively, our results have shown that majority of the Mᵖʳᵒ inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to Mᵖʳᵒ, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit Mᵖʳᵒ in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery.