Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

  1. Chunlong Ma
  2. Michael D. Sacco
  3. Brett Hurst
  4. Julia A. Townsend
  5. Yanmei Hu
  6. Tommy Szeto
  7. Xiujun Zhang
  8. Bart Tarbet
  9. Michael T. Marty
  10. Yu Chen
  11. Jun Wang

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Abstract

A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.96% as of May 4, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (M pro ). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC 50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC 50 values ranging from 0.49 to 3.37 μM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known M pro inhibitors. A complex crystal structure of SARS-CoV-2 M pro with GC-376, determined at 2.15 Å resolution with three monomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by M pro . Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.20.051581: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and viruses: Human rhabdomyosarcoma (RD); A549, MDCK, Caco-2, and Vero cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), BEAS2B and HCT-8 cells were maintained in RPMI 1640 medium.
    Caco-2
    suggested: None
    Vero
    suggested: None
    BEAS2B
    suggested: None
    Cytotoxicity measurement: A549, MDCK, HCT-8, Caco-2, Vero, and BEAS2B cells for cytotoxicity CPE assays were seeded and grown overnight at 37 °C in a 5% CO2 atmosphere to ~90% confluence on the next day.
    A549
    suggested: None
    MDCK
    suggested: None
    HCT-8
    suggested: None
    Briefly, supernatant virus was serially diluted in log10 increments then plated onto quadruplicate wells of 96-well plates seeded with Vero 76 cells.
    Vero 76
    suggested: IZSLER Cat# BS CL 101, RRID:CVCL_0603)
    Software and Algorithms
    SentencesResources
    The initial velocity was plotted against the FRET concentration with the classic Michaelis-Menten equation in Prism 5 software.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    The CC50 values were calculated from best-fit dose-response curves using GraphPad Prism 5 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Rigid and restrained refinements were performed using REFMAC and model building with COOT(10, 11).
    REFMAC
    suggested: (Refmac, RRID:SCR_014225)
    Protein structure figures were made using PyMOL (Schrödinger, LLC).
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    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 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.

    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 10.1101/2020.04.20.051581 on bioRxiv