Potent in vitro anti-SARS-CoV-2 activity by gallinamide A and analogues via inhibition of cathepsin L

  1. Anneliese S. Ashhurst
  2. Arthur H. Tang
  3. Pavla Fajtová
  4. Michael Yoon
  5. Anupriya Aggarwal
  6. Alexander Stoye
  7. Mark Larance
  8. Laura Beretta
  9. Aleksandra Drelich
  10. Danielle Skinner
  11. Linfeng Li
  12. Thomas D. Meek
  13. James H. McKerrow
  14. Vivian Hook
  15. Chien-Te K. Tseng
  16. Stuart Turville
  17. William H. Gerwick
  18. Anthony J. O’Donoghue
  19. Richard J. Payne

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Abstract

The emergence of SARS-CoV-2 in late 2019, and the subsequent COVID-19 pandemic, has led to substantial mortality, together with mass global disruption. There is an urgent need for novel antiviral drugs for therapeutic or prophylactic application. Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is recognized as a promising drug target. The marine natural product, gallinamide A and several synthetic analogues, were identified as potent inhibitors of cathepsin L activity with IC 50 values in the picomolar range. Lead molecules possessed selectivity over cathepsin B and other related human cathepsin proteases and did not exhibit inhibitory activity against viral proteases Mpro and PLpro. We demonstrate that gallinamide A and two lead analogues potently inhibit SARS-CoV-2 infection in vitro , with EC 50 values in the nanomolar range, thus further highlighting the potential of cathepsin L as a COVID-19 antiviral drug target.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.23.424111: (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
    MRC-5 cells stably expressing human ACE2 and TMPRSS2 were generated by transducing MRC-5 cells (ATCC CCL-171) with lentiviral particles.
    MRC-5
    suggested: None
    Lentiviral particles expressing the above proteins were produced by co-transfecting expression plasmids individually with a second generation lentiviral packaging construct psPAX2 (courtesy of Dr Didier Trono through NIH AIDS repository) and VSVG plasmid pMD2.G (Addgene#2259) in HEK293T cells (Life Technologies) by using polyethyleneimine as previously described (61).
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Human A549 cells stably transduced with human ACE2 viral receptor (A549/ACE2) were grown in M-10 medium.
    A549
    suggested: None
    The MRC5 and A549 cells were analysed using the Homo sapiens proteome database and the VeroE6 cells using the Chlorocebus sabaeus proteome database, both of which were from Uniprot.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    At the University of Texas Medical Branch, A549/ACE2 cells were grown in M-10 media and treated with 500 SARS-CoV-2 viral particles (USA_WA1/2020 isolate) prior to addition of 20 to 0.156 μM of compound.
    A549/ACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    To calculate Ki and kinact, the rate of product formation was calculated at 6 min intervals for 1 h and normalized to activity in the control wells containing DMSO. kobs values for each inhibitor concentration were calculated from inactivation curves, and inhibition constants kinact and kinact/Ki were calculated by nonlinear regression of kobs and inhibitor concentration using GraphPad Prism 9 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Identification and quantification were performed using MaxQuant with a 1% false discovery rate at the protein level using a target-decoy approach.
    MaxQuant
    suggested: (MaxQuant, RRID:SCR_014485)

    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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  2. Version published to 10.1101/2020.12.23.424111v1 on bioRxiv