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 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.

Challenges for Targeting SARS-CoV-2 Proteases as a Therapeutic Strategy for COVID-19

“…Interestingly, this compound was previously identified as a covalent inhibitor of SARS-CoV-1 M pro ( k inact / K I : 1900 ± 400 mol·s –1 ). 31 Following a recent report about the need for validation of M pro inhibitors under reducing conditions in order to exclude pan thiol-reactive compounds, 32 we verified our screening assay in the presence of reducing agent DTT in the assay buffer. Here we found that of the seven validated M pro hits, only JCP543 was partially sensitive to the reducing environment, losing approximately half of its inhibitory capacity in the presence of 4 mM DTT ( Figure S5 ).…”

An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity

“…Among the known M pro inhibitors, the majority of them are covalent inhibitors such as GC376 analogs that contain a pyrollidone in the P1 position as a glutamine mimetic. Several structurally distinct compounds including ebselen, disulfiram, carmofur, PX-12, tideglusib, and shiknonin were claimed as M pro inhibitors 16,17 , but were later invalidated as promiscuous non-specific cysteine protease inhibitors 18,19 . In addition, non-covalent inhibitors such as ML188 (R) were developed and validated as SARS-CoV M pro inhibitors 5,12 .…”

“…The expression and purification of SARS CoV-2 papain-like protease (PL pro ) was also described in our previous publications 7,8,18 .…”

Chinese herbal medicine: Fighting SARS-CoV-2 infection on all fronts

“…This study demonstrated different binding patterns, thus suggesting significant diversity in terms of binding sites. However, Ma et al recently revealed that shikonin might not be a target-specific SARS-CoV-2 M pro inhibitor, due to the fact that its inhibitory ability is greatly reduced in the presence of 1,4-dithiothreitol ( Ma et al, 2020a ). Our growing understanding of different binding modes has offered an important strategy for designing and identifying effective anti-SARS-CoV-2 agents.…”