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

Abstract

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 IC₅₀ 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.