Apixaban, an orally available anticoagulant, inhibits SARS-CoV-2 replication by targeting its major protease in a non-competitive way

  1. Otávio Augusto Chaves
  2. Carolina Q. Sacramento
  3. Natalia Fintelman-Rodrigues
  4. Jairo Ramos Temerozo
  5. Filipe Pereira-Dutra
  6. Daniella M. Mizurini
  7. Robson Q. Monteiro
  8. Leonardo Vazquez
  9. Patricia T. Bozza
  10. Hugo Caire Castro-Faria-Neto
  11. Thiago Moreno L. Souza

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Abstract

Anticoagulants are associated with clinical benefit against the 2019 coronavirus disease (COVID-19), preventing COVID-19 associated coagulopathy. Blood coagulation factor Xa (FXa) and SARS-CoV-2 major protease (M pro ) share over 80% homology at the three-dimensional protein level. Thus, it is worth interrogating whether there is crosstalk between inhibitors and substrates between these enzymes. Here, we found that the clinically-approved FXa inhibitor apixaban targets SARS-CoV-2 M pro with a 21-fold higher potency than boceprevir (GC376). Apixaban displayed a non-competitive mechanism of inhibition towards M pro , since it targets the enzyme/substrate complex and the allosteric site onto the viral protease. Enzymatic assays were further validated in infected Calu-3 cells, which reveal that apixaban decreases the production of infectious viral particles in a dose-dependent manner, with an inhibitory potency in the micromolar range. Our results are in line with the proposed early use of anticoagulants, including FXa inhibitors, to improve clinical outcome of COVID-19 patients. In this context, apixaban may display a dual mechanism of action by targeting FXa to prevent coagulopathy and, at some level, SARS-CoV-2 M pro .

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    SciScore for 10.1101/2021.09.23.461605: (What is this?)

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

    Table 1: Rigor

    EthicsEuthanasia Agents: In vitro assays: The Calu-3 cells were infected with multiplicity of infection (MOI) of 0.1 at densities of 2.0 × 105 cells/well for 1 h at 310K in 5 % of CO2.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    In vitro assays: The Calu-3 cells were infected with multiplicity of infection (MOI) of 0.1 at densities of 2.0 × 105 cells/well for 1 h at 310K in 5 % of CO2.
    Calu-3
    suggested: None
    After 48 h, the supernatants were harvested, and virus titers were quantified by plaque-based assays according to previous publications36-38, The cytotoxic assays were conducted in a monolayers of Vero cells (in about 2.0 × 104 cell/well) treated for 3 days with different concentrations of apixaban, rivaroxaban, dabigatran, atazanavir, or remdesivir (50, 150, 300, 600, and 800 µM) following procedure described by Sacramento, C.Q. et al37.
    Vero
    suggested: RRID:CVCL_ZW93)
    Software and Algorithms
    SentencesResources
    Statistics: All in vitro data were analyzed from Prism GraphPad software 8.0
    Prism GraphPad
    suggested: None
    Windows GraphPad Software, San Diego, California USA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The molecular docking calculations were performed with GOLD 2020.2 software (Cambridge Crystallographic Data Center Software Ltd., CCDC) at pH 7.4.
    GOLD
    suggested: (GOLD, RRID:SCR_000188)
    It was defined 8 Å radius around each main binding pockets and the figures of the best results were generated with PyMOL Delano Scientific LLC software (
    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 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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.09.23.461605v1 on bioRxiv