Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS-CoV-2

  1. Nir Drayman
  2. Jennifer K. DeMarco
  3. Krysten A. Jones
  4. Saara-Anne Azizi
  5. Heather M. Froggatt
  6. Kemin Tan
  7. Natalia Ivanovna Maltseva
  8. Siquan Chen
  9. Vlad Nicolaescu
  10. Steve Dvorkin
  11. Kevin Furlong
  12. Rahul S. Kathayat
  13. Mason R. Firpo
  14. Vincent Mastrodomenico
  15. Emily A. Bruce
  16. Madaline M. Schmidt
  17. Robert Jedrzejczak
  18. Miguel Á. Muñoz-Alía
  19. Brooke Schuster
  20. Vishnu Nair
  21. Kyu-yeon Han
  22. Amornrat O’Brien
  23. Anastasia Tomatsidou
  24. Bjoern Meyer
  25. Marco Vignuzzi
  26. Dominique Missiakas
  27. Jason W. Botten
  28. Christopher B. Brooke
  29. Hyun Lee
  30. Susan C. Baker
  31. Bryan C. Mounce
  32. Nicholas S. Heaton
  33. William E. Severson
  34. Kenneth E. Palmer
  35. Bryan C. Dickinson
  36. Andrzej Joachimiak
  37. Glenn Randall
  38. Savaş Tay

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Abstract

Inside host cells, the RNA genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is translated into two polyproteins that are cleaved to give the individual viral proteins. The main viral protease, known as Mpro or 3CLpro, plays a key role in these cleavages, making it an important drug target. Drayman et al . identified eight drugs that target 3CLpro from a library of 1900 clinically safe drugs. Because of the challenge of working with SARS-CoV-2, they started by screening for drugs that inhibit the replication of a human coronavirus that causes the common cold. They then evaluated the top hits for inhibiting SARS-CoV-2 replication and for inhibiting 3CLpro. Masitinib, a broad antiviral, inhibited the main proteases of coronaviruses and picornaviruses and was effective in reducing SARS-CoV-2 replication in mice. —VV

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  1. Version published to 10.1126/science.abg5827
  2. 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

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  3. Version published to 10.1101/2020.08.31.274639v1 on bioRxiv