Ebselen, disulfiram, carmofur, PX-12, tideglusib, and shikonin are non-specific promiscuous SARS-CoV-2 main protease inhibitors
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Abstract
There is an urgent need for vaccines and antiviral drugs to combat the COVID-19 pandemic. Encouraging progress has been made in developing antivirals targeting SARS-CoV-2, the etiological agent of COVID-19. Among the drug targets being investigated, the viral main protease (M pro ) is one of the most extensively studied drug targets. M pro is a cysteine protease that hydrolyzes the viral polyprotein at more than 11 sites and it is highly conserved among coronaviruses. In addition, M pro has a unique substrate preference for glutamine in the P1 position. Taken together, it appears that M pro inhibitors can achieve both broad-spectrum antiviral activity and a high selectivity index. Structurally diverse compounds have been reported as M pro inhibitors, with several of which also showed antiviral activity in cell culture. 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 non-specific and the inhibition is abolished or greatly reduced with the addition of reducing reagent DTT. In the absence of DTT, these six compounds not only inhibit M pro , but also a panel of viral cysteine proteases including SARS-CoV-2 papain-like protease, the 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 ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 are non-specific SARS-CoV-2 M pro inhibitors, and urge the scientific community to be stringent with hit validation.
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SciScore for 10.1101/2020.09.15.299164: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources 39 Cytopathic Effect Assay (CPE): The EC50 and CC50 values for the protease inhibitors investigated in this study were measured using RD cells as described previously.32 Briefly, RD cells were seeded and grown overnight to ∼90% confluence in 96-well plate at 37 °C 5% CO2 incubator. RDsuggested: NoneExperimental Models: Organisms/Strains Sentences Resources EV-A71 2Apro: EV-A71 2Apro gene from strain EV-A71/7D3 (genbank accession number … SciScore for 10.1101/2020.09.15.299164: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources 39 Cytopathic Effect Assay (CPE): The EC50 and CC50 values for the protease inhibitors investigated in this study were measured using RD cells as described previously.32 Briefly, RD cells were seeded and grown overnight to ∼90% confluence in 96-well plate at 37 °C 5% CO2 incubator. RDsuggested: NoneExperimental Models: Organisms/Strains Sentences Resources EV-A71 2Apro: EV-A71 2Apro gene from strain EV-A71/7D3 (genbank accession number MF973167) with E. coli codon optimization was ordered from GenScript in the pET28b(+) vector. EV-A71 2Apro: EV-A71 2Aprosuggested: NoneEV-A71 3Cpro: EV-A71 3Cpro gene from strain EV-A71/7D3 (genbank accession number MF973167) with E. coli codon optimization was ordered from GenScript in the pET28b(+) vector. EV-A71 3Cpro: EV-A71 3Cprosuggested: NoneEV-A71 3Cpro substrate: Dabcyl-IEALFQ/GPPKFRE-Edan EV-D68 2Apro substrate: Dabcyl-KIRIVNT/GPGFGGE-Edan EV-D68 3Cpro substrate: Dabcyl-KEALFQ/GPPQFE-Edans The synthesis of SARS-CoV-2 Mpro, PLpro, EV-A71 2Apro, EV-D68 2Apro and EV-D68 3Cpro substrates were described previously.11, 33 Enzymatic assays: The IC50 values of the testing compounds against various SARS-CoV-2, EV-A71 and EV-D68 proteases in the presence or in the absence of 4 mM DTT were measured with a common protocol as the following: 100 µl protease (SARS-CoV-2 Mpro at 100 nM; SARS-CoV-2 PLpro at 200 nM; EV-A71 2Apro at 3 µM; EV-A71 3Cpro at 2 µM, EV-D68 2Apro at 1 µM, or EV-D68 3Cpro at 100 nM) was incubated with various concentrations of testing inhibitors at 30°C for 30 min in its reaction buffer in 96-well plate, and then the reaction was initiated by adding FRET substrate (SARS-CoV-2 Mpro and PLpro substrates at 10 µM; and EV-A71 and EV-D68 substrates at 20 µM), the reaction was monitored for 2□h, and the initial velocity was calculated using the data from the first 15□min by linear regression. EV-A71 3Cpro substrate: Dabcyl-IEALFQ/GPPKFRE-Edan EV-D68 2Apro substrate: Dabcyl-KIRIVNT/GPGFGGE-Edan EV-D68 3Cprosuggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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