Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease

  1. Jennifer C. Milligan
  2. Theresa U. Zeisner
  3. George Papageorgiou
  4. Dhira Joshi
  5. Christelle Soudy
  6. Rachel Ulferts
  7. Mary Wu
  8. Chew Theng Lim
  9. Kang Wei Tan
  10. Florian Weissmann
  11. Berta Canal
  12. Ryo Fujisawa
  13. Tom Deegan
  14. Hema Nagaraj
  15. Ganka Bineva-Todd
  16. Clovis Basier
  17. Joseph F. Curran
  18. Michael Howell
  19. Rupert Beale
  20. Karim Labib
  21. Nicola O'Reilly
  22. John F.X. Diffley

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Abstract

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

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  1. Version published to 10.1042/bcj20210197
  2. ScreenIT

    SciScore for 10.1101/2021.04.07.438806: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    The conditions for the viral inhibition assay in Vero E6 cells are described in Zheng et al (Biochem J, this issue).
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Identification of Primary Hits: Time-course data was analysed using MATLAB (R2020_a) to determine the rate of reaction then normalised to the rate of reaction of positive (no drug compound) control reaction wells.
    MATLAB
    suggested: (MATLAB, RRID:SCR_001622)
    IC50 values were then calculated using non-linear regression of inhibitor concentration against the normalised response using variable slope via GraphPad Prism (Version 8.4.3)
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 10.1101/2021.04.07.438806v1 on bioRxiv