Ebselen, disulfiram, carmofur, PX-12, tideglusib, and shikonin are non-specific promiscuous SARS-CoV-2 main protease inhibitors

Ma, Chunlong; Hu, Yanmei; Townsend, Julia Alma; Lagarias, Panagiotis; Marty, Michael T.; Kolocouris, Antonios; Wang, Jun

doi:10.1101/2020.09.15.299164

Cited by 20 publications

(27 citation statements)

References 42 publications

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“…In general, we observe concordance between compounds showing activity within this transfection-based 3CLpro assay and live virus studies (Supplementary Fig. 4a-e) [14,30]. However, within our assay we did not observe activity for ebselen, a small molecule with demonstrated in vitro activity against purified SARSpg.…”

Section: Compound Rescue Of Transfected 3clpro Cytotoxicity Mimics the Results Obtained With Live Virussupporting

confidence: 77%

Inhibitors of Coronavirus 3CL Proteases Protect Cells from Protease-Mediated Cytotoxicity

Resnick

Iketani

Hong

et al. 2021

J Virol

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We describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This assay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, we identify compounds with broad 3CLpro inhibitory activity. We also adapt the assay for use in compound screening and in doing so uncover additional SARS-CoV-2 3CLpro inhibitors. We observe strong concordance between data emerging from this assay and those obtained from live virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. We identify two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested including 3CLpro enzymes from understudied zoonotic coronaviruses. Importance Multiple coronavirus pandemics have occurred over the last two decades. This has highlighted a need to be proactive in the development of therapeutics that can be readily deployed in the case of future coronavirus pandemics. We develop and validate a simplified cell-based assay for the identification of chemical inhibitors of 3CL proteases encoded by a wide range of coronaviruses. This assay is reporter-free, does not require specialized biocontainment, and is optimized for performance in high-throughput screening. By testing reported 3CL protease inhibitors against a large collection of 3CL proteases with variable sequence similarity, we identify compounds with broad activity against 3CL proteases and uncover structural insights that contribute to their broad activity. Furthermore, we demonstrate this assay is suitable for identifying chemical inhibitors of proteases from families other than 3CL proteases.

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Section: Compound Rescue Of Transfected 3clpro Cytotoxicity Mimics the Results Obtained With Live Virussupporting

confidence: 77%

Inhibitors of Coronavirus 3CL Proteases Protect Cells from Protease-Mediated Cytotoxicity

Resnick

Iketani

Hong

et al. 2021

J Virol

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“…Since 1951, Disulfiram is used in the treatment of alcohol aversion and is also a competitive inhibitor for SARS-CoV Plpro. 64 With another papain-like protease M pro phytochemical Germacranolide showed the most potential interaction during molecular docking studies in comparison to that of its control molecule (Lopinavir).…”

Section: Resultsmentioning

confidence: 99%

The dual role of phytochemicals on SARS-CoV-2 inhibition by targeting host and viral proteins

Singh

Chauhan

Pandit

et al. 2022

Journal of Traditional and Complementary Medicine

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Background The severe acute respiratory syndrome-2019 has affected more than 190 million people around the world and caused severe crises throughout the globe. Due to rapid mutation in the viral genome, it became important to simultaneously improvise the host immunity while targeting the viral protein to reduce the severity of the infection. Aim The current computational work focuses on multi-level rigorous screening of 47 medicinal plant-based phytochemicals for discovering effective phytochemical inhibitors against the host and viral targets. Experimental procedure A total of 586 phytochemicals were analyzed in detail based on their drug-likeness, pharmacological properties, and structure-based activity against the viral proteins (Spike glycoprotein, Papain-like protease, and Main protease) and host proteins (ACE2, Importin-subunit α-5, and β-1). Phytochemicals showing higher binding affinity with the dual capacity to target both the categories of proteins were further analyzed by profiling their chemical reactivity using Density-functional theory (DFT) based quantum chemical methods. Finally, detailed molecular dynamics simulations were performed to analyze the interactions of the complexes. Results and conclusion The results revealed that the selected phytochemicals from Andrographis paniculata , Aconitum heterophyllum , Costus speciosus and Inula racemosa may have the capacity to act with prominent affinity towards the host and viral proteins. Therefore, The Combination of active phytochemicals of these plants may prove to be more beneficial and can be used for developing the potential phytotherapeutic intervention.

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“…Our findings suggest that they do not, and that their possible antiviral activity may have been an artifact due to cytotoxicity, as described for lopinavir and nelfinavir [ 38 ]. Although most of these compounds were reported to inhibit 3CL pro in vitro, they may have done so through non-specific mechanisms, as recently reported for ebselen [ 59 ]. Second, the compounds may not be toxic but still fail to inhibit 3CL pro in cells due to poor permeability, metabolization into an inactive form, or a lack of specificity for 3CL pro .…”

Section: Discussionmentioning

confidence: 95%

Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors

Rawson

Duchon

Nikolaitchik

et al. 2021

Viruses

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The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.

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Ebselen, disulfiram, carmofur, PX-12, tideglusib, and shikonin are non-specific promiscuous SARS-CoV-2 main protease inhibitors

Cited by 20 publications

References 42 publications

Inhibitors of Coronavirus 3CL Proteases Protect Cells from Protease-Mediated Cytotoxicity

Inhibitors of Coronavirus 3CL Proteases Protect Cells from Protease-Mediated Cytotoxicity

The dual role of phytochemicals on SARS-CoV-2 inhibition by targeting host and viral proteins

Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors

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