Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays

SARS-CoV-2 main protease (M ^pro ) is one of the most extensive exploited drug targets for COVID-19. Structurally disparate compounds have been reported as M ^pro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed M ^pro 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 Flip-GFP assay. Collectively, our results have shown that majority of the M ^pro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to M ^pro , while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit M ^pro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery.