Characterization of raloxifene as a potential pharmacological agent against SARS-CoV-2 and its variants

  1. Daniela Iaconis
  2. Licia Bordi
  3. Giulia Matusali
  4. Carmine Talarico
  5. Candida Manelfi
  6. Maria Candida Cesta
  7. Mara Zippoli
  8. Francesca Caccuri
  9. Antonella Bugatti
  10. Alberto Zani
  11. Federica Filippini
  12. Laura Scorzolini
  13. Marco Gobbi
  14. Marten Beeg
  15. Arianna Piotti
  16. Monica Montopoli
  17. Veronica Cocetta
  18. Silvia Bressan
  19. Enrico M. Bucci
  20. Arnaldo Caruso
  21. Emanuele Nicastri
  22. Marcello Allegretti
  23. Andrea R. Beccari

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Abstract

The new coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic, which so far has caused over 6 million deaths in 2 years, despite new vaccines and antiviral medications. Drug repurposing, an approach for the potential application of existing pharmaceutical products to new therapeutic indications, could be an effective strategy to obtain quick answers to medical emergencies. Following a virtual screening campaign on the most relevant viral proteins, we identified the drug raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a new potential agent to treat mild-to-moderate COVID-19 patients. In this paper we report a comprehensive pharmacological characterization of raloxifene in relevant in vitro models of COVID-19, specifically in Vero E6 and Calu-3 cell lines infected with SARS-CoV-2. A large panel of the most common SARS-CoV-2 variants isolated in Europe, United Kingdom, Brazil, South Africa and India was tested to demonstrate the drug’s ability in contrasting the viral cytopathic effect (CPE). Literature data support a beneficial effect by raloxifene against the viral infection due to its ability to interact with viral proteins and activate protective estrogen receptor-mediated mechanisms in the host cells. Mechanistic studies here reported confirm the significant affinity of raloxifene for the Spike protein, as predicted by in silico studies, and show that the drug treatment does not directly affect Spike/ACE2 interaction or viral internalization in infected cell lines. Interestingly, raloxifene can counteract Spike-mediated ADAM17 activation in human pulmonary cells, thus providing new insights on its mechanism of action. A clinical study in mild to moderate COVID-19 patients (NCT05172050) has been recently completed. Our contribution to evaluate raloxifene results on SARS-CoV-2 variants, and the interpretation of the mechanisms of action will be key elements to better understand the trial results, and to design new clinical studies aiming to evaluate the potential development of raloxifene in this indication.

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  1. Version published to 10.1038/s41419-022-04961-z
  2. ScreenIT

    SciScore for 10.1101/2021.10.22.465294: (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
    After being blocked with 3% BSA in TBS buffer containing 0.05% Tween 20, the blot was probed with a human serum (1:1000 dilution) containing IgG to the SARS-CoV-2 nucleoprotein (NP) and with mouse anti-human GAPDH monoclonal antibody (G-9: Santa Cruz Biotechnology, Dallas, TX, USA).
    SARS-CoV-2 nucleoprotein (NP)
    suggested: None
    anti-human GAPDH
    suggested: None
    The antigen-antibody complexes were detected using peroxidase-conjugated goat anti-human or goat anti-mouse IgG (Sigma) and revealed using the enhanced chemiluminescence (ECL) system (Santa Cruz Biotechnology).
    anti-human or goat anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Subsequently, we screened 8721 Scopus-derived documents, referred to raloxifene, for the presence of at least one of the proteins/genes included in the Cytoscape-generated network; this allowed to isolate 600 papers, which were manually examined and annotated for enriched human gene ontology according to BiNGO v.3.5.0. Cells: African green monkey kidney Vero E6 cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle Medium
    Vero E6
    suggested: RRID:CVCL_XD71)
    Calu-3 (human, Caucasian, lung, adenocarcinoma) cell line was obtained from ATCC and maintained in Minimum Essential Medium (MEM; Gibco, Thermo-Fisher) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo-Fisher) at 37°C in a humidified atmosphere of 5% CO2.
    Calu-3
    suggested: None
    Recombinant DNA
    SentencesResources
    A standard curve was generated by determination of copy numbers derived from serial dilutions (103-109 copies) of a pGEM T-easy vector (Promega, Madison, WI, USA) containing the receptor binding domain of the S gene (primers: RBD-F: 5’-GCTGGATCCCCTAATATTACAAACTTGTGCC-3’; RBD-R: 5’-TGCCTCGAGCTCAAGTGTCTGTGGATCAC-3’).
    pGEM T-easy
    suggested: None
    Software and Algorithms
    SentencesResources
    A list combining the two dataset was used as seeding for a BioGrid search by mean of Cytoscape v.
    BioGrid
    suggested: (BioGrid Australia, RRID:SCR_006334)
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    Subsequently, we screened 8721 Scopus-derived documents, referred to raloxifene, for the presence of at least one of the proteins/genes included in the Cytoscape-generated network; this allowed to isolate 600 papers, which were manually examined and annotated for enriched human gene ontology according to BiNGO v.3.5.0. Cells: African green monkey kidney Vero E6 cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle Medium
    BiNGO
    suggested: None
    Data analysis: The half-cytotoxic concentration (CC50) and the half-maximal inhibitory concentration (IC50) for raloxifene were calculated from concentration-effect-curves after non-linear regression analysis using GraphPad Prism8.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Statistical tests were performed using GraphPad Prism 8.
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04388683TerminatedInhaled Nitric Oxide for Preventing Progression in COVID-19

    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.
    • No funding statement was detected.
    • 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 10.1101/2021.10.22.465294v1 on bioRxiv