Ebselen, Disulfiram, Carmofur, PX-12, Tideglusib, and Shikonin Are Nonspecific Promiscuous SARS-CoV-2 Main Protease Inhibitors

[Image: see text] Among the drug targets being investigated for SARS-CoV-2, the viral main protease (M(pro)) is one of the most extensively studied. M(pro) is a cysteine protease that hydrolyzes the viral polyprotein at more than 11 sites. It is highly conserved and has a unique substrate preference for glutamine in the P1 position. Therefore, M(pro) inhibitors are expected to have broad-spectrum antiviral activity and a high selectivity index. Structurally diverse compounds have been reported as M(pro) inhibitors. In this study, we investigated the mechanism of action of six previously reported M(pro) inhibitors, ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12, using a consortium of techniques including FRET-based enzymatic assay, thermal shift assay, native mass spectrometry, cellular antiviral assays, and molecular dynamics simulations. Collectively, the results showed that the inhibition of M(pro) by these six compounds is nonspecific and that the inhibition is abolished or greatly reduced with the addition of reducing reagent 1,4-dithiothreitol (DTT). Without DTT, these six compounds inhibit not only M(pro) but also a panel of viral cysteine proteases including SARS-CoV-2 papain-like protease and 2A(pro) and 3C(pro) from enterovirus A71 (EV-A71) and EV-D68. However, none of the compounds inhibits the viral replication of EV-A71 or EV-D68, suggesting that the enzymatic inhibition potency IC(50) values obtained in the absence of DTT cannot be used to faithfully predict their cellular antiviral activity. Overall, we provide compelling evidence suggesting that these six compounds are nonspecific SARS-CoV-2 M(pro) inhibitors and urge the scientific community to be stringent with hit validation.