Drug repurposing screen identifies masitinib as a 3CLpro inhibitor that blocks replication of SARS-CoV-2 in vitro

  1. Nir Drayman
  2. Krysten A. Jones
  3. Saara-Anne Azizi
  4. Heather M. Froggatt
  5. Kemin Tan
  6. Natalia Ivanovna Maltseva
  7. Siquan Chen
  8. Vlad Nicolaescu
  9. Steve Dvorkin
  10. Kevin Furlong
  11. Rahul S. Kathayat
  12. Mason R. Firpo
  13. Vincent Mastrodomenico
  14. Emily A. Bruce
  15. Madaline M. Schmidt
  16. Robert Jedrzejczak
  17. Miguel Á. Muñoz-Alía
  18. Brooke Schuster
  19. Vishnu Nair
  20. Jason W. Botten
  21. Christopher B. Brooke
  22. Susan C. Baker
  23. Bryan C. Mounce
  24. Nicholas S. Heaton
  25. Bryan C. Dickinson
  26. Andrzej Jaochimiak
  27. Glenn Randall
  28. Savaş Tay

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Abstract

There is an urgent need for anti-viral agents that treat SARS-CoV-2 infection. The shortest path to clinical use is repurposing of drugs that have an established safety profile in humans. Here, we first screened a library of 1,900 clinically safe drugs for inhibiting replication of OC43, a human beta-coronavirus that causes the common-cold and is a relative of SARS-CoV-2, and identified 108 effective drugs. We further evaluated the top 26 hits and determined their ability to inhibit SARS-CoV-2, as well as other pathogenic RNA viruses. 20 of the 26 drugs significantly inhibited SARS-CoV-2 replication in human lung cells (A549 epithelial cell line), with EC50 values ranging from 0.1 to 8 micromolar. We investigated the mechanism of action for these and found that masitinib, a drug originally developed as a tyrosine-kinase inhibitor for cancer treatment, strongly inhibited the activity of the SARS-CoV-2 main protease 3CLpro. X-ray crystallography revealed that masitinib directly binds to the active site of 3CLpro, thereby blocking its enzymatic activity. Mastinib also inhibited the related viral protease of picornaviruses and blocked picornaviruses replication. Thus, our results show that masitinib has broad anti-viral activity against two distinct beta-coronaviruses and multiple picornaviruses that cause human disease and is a strong candidate for clinical trials to treat SARS-CoV-2 infection.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.31.274639: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    BlindingSpike positive cells (n>40) were quantified by light microscopy as blinded samples.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After 48 hours, the cells were fixed using 3.7% formalin, blocked and probed with mouse anti-Spike antibody (GTX632604, GeneTex) diluted 1:1,000 for 4 hours, rinsed and probed with anti-mouse-HRP for 1 hour, washed, then developed with DAB substrate 10 minutes.
    anti-Spike
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    GTX632604
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    anti-mouse-HRP
    suggested: (Kindle Biosciences Cat# R1005, RRID:AB_2800463)
    Experimental Models: Cell Lines
    SentencesResources
    Cells: A549 expressing H2B-mRuby were generated by first infecting A549 cells (ATCC CCL-185) with a lentivirus (carrying H2B-mRuby), and FACS-sorting mRuby+ cells.
    A549
    suggested: None
    We used Huh7 cells for picornaviruses infections.
    Huh7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    MDCK-SIAT1-TMPRSS2 cells, obtained from Jesse Bloom, were used for IAV infections.
    MDCK-SIAT1-TMPRSS2
    suggested: None
    CVB3 (Nancy strain), HRV 2, 14, and 16 were derived from full-length infectious clones and generated in Vero cells (NR-10385, BEI Resources, NIAID, NIH).
    Vero
    suggested: None
    Working stocks were generated in Vero E6 cells, and the same cells were used to measure virus titers.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Drug screening: A549-mRuby cells were seeded (3,000 cells per well) in nine 384-well plates using Multidrop combi.
    A549-mRuby
    suggested: None
    Ace2-A549 cells in DMEM +2% FBS were treated with drugs for 2 hours with 2-fold dilutions beginning at 10μM in triplicate for each assay.
    Ace2-A549
    suggested: None
    FlipGFP SARS-CoV-2 3CLpro Assay: 293T cells were seeded 24 hours before transfection on poly-lysine treated plates.
    293T
    suggested: None
    Software and Algorithms
    SentencesResources
    A sigmoid fit was used to extract EC50 values using Matlab.
    Matlab
    suggested: (MATLAB, RRID:SCR_001622)
    10 kDa MWCO filter (Amicon-Millipore) was used to concentrate the protein solution, which was subsequently applied to Superdex 75 column, pre-equilibrated with lysis buffer.
    Amicon-Millipore
    suggested: None

    Results from OddPub: Thank you for sharing your data.

    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 did not find any issues relating to the usage of bar graphs.

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.08.31.274639 on bioRxiv