Mitoxantrone modulates a glycosaminoglycan-spike complex to inhibit SARS-CoV-2 infection

  1. Qi Zhang
  2. Peter Radvak
  3. Juhyung Lee
  4. Yue Xu
  5. Vivian Cao-Dao
  6. Miao Xu
  7. Wei Zheng
  8. Catherine Z. Chen
  9. Hang Xie
  10. Yihong Ye

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Abstract

Spike-mediated entry of SARS-CoV-2 into human airway epithelial cells is an attractive therapeutic target for COVID-19. In addition to protein receptors, the SARS-CoV-2 spike (S) protein also interacts with heparan sulfate, a negatively charged glycosaminoglycan (GAG) attached to certain membrane proteins on the cell surface. This interaction facilitates the engagement of spike with a downstream receptor to promote viral entry. Here, we show that Mitoxantrone, an FDA-approved topoisomerase inhibitor, targets a spike-GAG complex to compromise the fusogenic function of spike in viral entry. As a single agent, Mitoxantrone inhibits the infection of an authentic SARS-CoV-2 strain in a cell-based model and in human lung EpiAirway 3D tissues. Gene expression profiling supports the plasma membrane as a major target of Mitoxantrone but also underscores an undesired activity targeting nucleosome dynamics. We propose that Mitoxantrone analogs bearing similar GAG-binding activities but with reduced affinity for DNA topoisomerase may offer an alternative therapy to overcome breakthrough infections in the post-vaccine era.

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    SciScore for 10.1101/2021.10.15.464595: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All live virus infections were performed in a biosafety level 3 (BSL3) laboratory as approved by the Institutional Biosafety Committee (IBC).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The viral spike protein was stained with an in-house developed rabbit anti-spike antibody and goat anti-rabbit IgG (H+L) highly cross-adsorbed antibody with Alexa Fluor 488 (Invitrogen).
    anti-spike
    suggested: None
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Effect cells were HEK293T cells stably expressing ACE2-GFP, as described previously 21, or as a control, HEK293T cells transfected with pEGFP.
    HEK293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    SARS-CoV-2 infection on cells: 7×104 Vero E6 cells (ATCC #CRL-1586) were seeded into an Millicell EZ SLIDES chamber slides (Millipore) 24 hours before infection.
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    pcDNA3.1-SARS-CoV-2-Spike plasmid was obtained from BEI resource 7. pLV-Mcherry was obtained from Addgene (Catalog number:
    pcDNA3.1-SARS-CoV-2-Spike
    suggested: None
    pLV-Mcherry
    suggested: RRID:Addgene_36084)
    Effect cells were HEK293T cells stably expressing ACE2-GFP, as described previously 21, or as a control, HEK293T cells transfected with pEGFP.
    pEGFP
    suggested: RRID:Addgene_165830)
    Software and Algorithms
    SentencesResources
    The identified networks were exported into Cytoscape for graph-making.
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    To determine how Mitoxantrone interacts with DNA and topoisomerase, we used LigPlot to analyze the molecular interactions present in the published structure (PDB: 4g0v) 27.
    LigPlot
    suggested: (LigPlot+, RRID:SCR_018249)
    Image processing and statistical analyses: Confocal images were processed using the Zeiss Zen software.
    Zeiss Zen
    suggested: None
    To measure fluorescence intensity, we used the open-source Fiji software.
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Statistical analyses were performed using either Excel or GraphPad Prism 9.
    Excel
    suggested: None
    Data are presented as means□±□SEM, which was calculated by GraphPad Prism 9.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Images were prepared with Adobe Photoshop and assembled in Adobe Illustrator.
    Adobe Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)
    Adobe Illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)

    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: 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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 19 and 22. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2021.10.15.464595v1 on bioRxiv