Common cardiac medications potently inhibit ACE2 binding to the SARS-CoV-2 Spike, and block virus penetration and infectivity in human lung cells

  1. Hung Caohuy
  2. Ofer Eidelman
  3. Tinghua Chen
  4. Shufeng Liu
  5. Qingfeng Yang
  6. Alakesh Bera
  7. Nathan I. Walton
  8. Tony T. Wang
  9. Harvey B. Pollard

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Abstract

To initiate SARS-CoV-2 infection, the Receptor Binding Domain (RBD) on the viral spike protein must first bind to the host receptor ACE2 protein on pulmonary and other ACE2-expressing cells. We hypothesized that cardiac glycoside drugs might block the binding reaction between ACE2 and the Spike (S) protein, and thus block viral penetration into target cells. To test this hypothesis we developed a biochemical assay for ACE2:Spike binding, and tested cardiac glycosides as inhibitors of binding. Here we report that ouabain, digitoxin, and digoxin, as well as sugar-free derivatives digitoxigenin and digoxigenin, are high-affinity competitive inhibitors of ACE2 binding to the Original [D614] S1 and the α/β/γ [D614G] S1 proteins. These drugs also inhibit ACE2 binding to the Original RBD, as well as to RBD proteins containing the β [E484K], Mink [Y453F] and α/β/γ [N501Y] mutations. As hypothesized, we also found that ouabain, digitoxin and digoxin blocked penetration by SARS-CoV-2 Spike-pseudotyped virus into human lung cells, and infectivity by native SARS-CoV-2. These data indicate that cardiac glycosides may block viral penetration into the target cell by first inhibiting ACE2:RBD binding. Clinical concentrations of ouabain and digitoxin are relatively safe for short term use for subjects with normal hearts. It has therefore not escaped our attention that these common cardiac medications could be deployed worldwide as inexpensive repurposed drugs for anti-COVID-19 therapy.

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  1. Version published to 10.1038/s41598-021-01690-9
  2. ScreenIT

    SciScore for 10.1101/2021.06.02.446343: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    A working concentration of 1 μg/ml human ACE2 biotinylated antibody (R&D Systems, Minneapolis, MN) was prepared, and 100 μL of this solution was added to each well.
    ACE2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Recombinant proteins produced in human T293 cells were obtained as follows: Recombinant Human Angiotensin Converting Enzyme 2 (ACE2) (Cat # 230-30165; lot 04U06020TW) was purchased from RayBiotech (Peachtree Corners, GA,
    T293
    suggested: RRID:CVCL_T293)
    Thereafter, 100 μL of the mixture was transferred to a semi-confluent culture of A549 human alveolar basal epithelial cells growing at a density of 20,000 cells/well in a 96-well white cell culture plate with a flat clear bottom.
    A549
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistics: To determine the significance of changes in kinetic parameters of ACE2 binding to SARS-CoV-2 Spike or RBD mutants we have applied least-squares regression to the linearized Eadie-Hoffstee plot of the binding data, and determined the statistical significance of the difference between the slopes (i.e., KD’s) and between the intercepts (i.e., Bmax) using R or Stata statistical packages.
    Stata
    suggested: (Stata, RRID:SCR_012763)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    It is a limitation of our study that we have not tested whether changes in spike conformation in response to ACE2 binding might also be occurring. Certainly infectivity enhancing mutations such as Mink [Y453F], UK [N501Y] and S.Africa [E484K] profoundly affect ACE2 binding kinetics. But whether RBD conformation intrinsically changes in response to ACE2 binding is a study for the future. An additional limitation of our study is that we have utilized a luc-loaded, SARS-CoV-2 pseudotyped virus as a test for biological activity rather than a native SARS-CoV-2 virus. However, it was only with a viral particle model that we could unambiguously demonstrate that cardiac glycosides inhibited viral entry. As mentioned above, ouabain itself has also been previously demonstrated to block infectivity by native SARS-CoV-2 virus, albeit in a green monkey Vero cell 22. Conclusion: Ouabain and digitoxin competitively inhibit ACE2 binding to the SARS-CoV-2 Receptor Binding Domain (RBD), and also block virus penetration into human lung cells. Clinical concentrations of cardiac glycosides are relatively safe for subjects with normal hearts, and it is therefore possible that these drugs could be repurposed for COVID-19 therapy.

    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.

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

    About SciScore

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  3. Version published to 10.1101/2021.06.02.446343v1 on bioRxiv