Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease

  1. Berta Canal
  2. Ryo Fujisawa
  3. Allison W. McClure
  4. Tom D. Deegan
  5. Mary Wu
  6. Rachel Ulferts
  7. Florian Weissmann
  8. Lucy S. Drury
  9. Agustina P. Bertolin
  10. Jingkun Zeng
  11. Rupert Beale
  12. Michael Howell
  13. Karim Labib
  14. John F.X. Diffley

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Abstract

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.

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  1. Version published to 10.1042/bcj20210199
  2. ScreenIT

    SciScore for 10.1101/2021.04.07.438811: (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

    Antibodies
    SentencesResources
    22 h post-infection, cells were fixed, permeabilised and stained for SARS-CoV-2 N-protein using Alexa488-labelled-CR3009 antibody [53] and nuclei using DRAQ5 (647 nm wavelength).
    DRAQ5
    suggested: (Biostatus Cat# DR50050, RRID:AB_2314341)
    Experimental Models: Cell Lines
    SentencesResources
    Baculoviruses were generated and amplified in Sf9 cells (Thermo Fisher Scientific) using the EMBacY baculoviral genome (Trowitzsch et al., J Struct Biol, 2010).
    Sf9
    suggested: None
    Briefly, VERO E6 cells were grown in 96 well imaging plates and transfected with individual gapmers at 0.5 μM using Lipofectamine 2000 (Thermofisher).
    VERO E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Percent activity for each log10 of the concentration of NSC95397 (μM) were then used to estimate the IC50 and Hill slopes of NSC95397 for nsp15 endoribonuclease using GraphPad Prism.
    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 10.1101/2021.04.07.438811v1 on bioRxiv