Unlike Chloroquine, Mefloquine Inhibits SARS-CoV-2 Infection in Physiologically Relevant Cells

  1. Carolina Q. Sacramento
  2. Natalia Fintelman-Rodrigues
  3. Suelen S. G. Dias
  4. Jairo R. Temerozo
  5. Aline de Paula D. Da Silva
  6. Carine S. da Silva
  7. Camilla Blanco
  8. André C. Ferreira
  9. Mayara Mattos
  10. Vinicius C. Soares
  11. Filipe Pereira-Dutra
  12. Milene Dias Miranda
  13. Debora F. Barreto-Vieira
  14. Marcos Alexandre N. da Silva
  15. Suzana S. Santos
  16. Mateo Torres
  17. Otávio Augusto Chaves
  18. Rajith K. R. Rajoli
  19. Alberto Paccanaro
  20. Andrew Owen
  21. Dumith Chequer Bou-Habib
  22. Patrícia T. Bozza
  23. Thiago Moreno L. Souza

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Abstract

Despite the development of specific therapies against severe acute respiratory coronavirus 2 (SARS-CoV-2), the continuous investigation of the mechanism of action of clinically approved drugs could provide new information on the druggable steps of virus–host interaction. For example, chloroquine (CQ)/hydroxychloroquine (HCQ) lacks in vitro activity against SARS-CoV-2 in TMPRSS2-expressing cells, such as human pneumocyte cell line Calu-3, and likewise, failed to show clinical benefit in the Solidarity and Recovery clinical trials. Another antimalarial drug, mefloquine, which is not a 4-aminoquinoline like CQ/HCQ, has emerged as a potential anti-SARS-CoV-2 antiviral in vitro and has also been previously repurposed for respiratory diseases. Here, we investigated the anti-SARS-CoV-2 mechanism of action of mefloquine in cells relevant for the physiopathology of COVID-19, such as Calu-3 cells (that recapitulate type II pneumocytes) and monocytes. Molecular pathways modulated by mefloquine were assessed by differential expression analysis, and confirmed by biological assays. A PBPK model was developed to assess mefloquine’s optimal doses for achieving therapeutic concentrations. Mefloquine inhibited SARS-CoV-2 replication in Calu-3, with an EC50 of 1.2 µM and EC90 of 5.3 µM. It reduced SARS-CoV-2 RNA levels in monocytes and prevented virus-induced enhancement of IL-6 and TNF-α. Mefloquine reduced SARS-CoV-2 entry and synergized with Remdesivir. Mefloquine’s pharmacological parameters are consistent with its plasma exposure in humans and its tissue-to-plasma predicted coefficient points suggesting that mefloquine may accumulate in the lungs. Altogether, our data indicate that mefloquine’s chemical structure could represent an orally available host-acting agent to inhibit virus entry.

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  1. Version published to 10.3390/v14020374
  2. ScreenIT

    SciScore for 10.1101/2021.07.21.451321: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    The purity of human monocytes was above 95%, as determined by flow cytometric analysis (FACScan; Becton Dickinson) using anti-CD3 (BD Biosciences) and anti-CD16 (Southern Biotech) monoclonal antibodies.
    anti-CD3
    suggested: None
    anti-CD16
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Vero-E6 cells were infected with SARS-CoV-2 at MOI of 0.01.
    Vero-E6
    suggested: None
    To evaluate the effects of mefloquine on SARS-CoV-2 attachment, monolayers of Calu-3 cells in 96-well plates (2 × 105 cells/well) were infected with MOI of 0.1, for 1h at 37°C.
    Calu-3
    suggested: None
    Among the different cell lines available in CMAP, we chose data from A549 cells, which is the one most similar to Calu-3 used in our wet lab experiments.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    Software and Algorithms
    SentencesResources
    Remdesevir (RDV), was purchased from Selleckhem (https://www.selleckchem.com/).
    https://www.selleckchem.com/
    suggested: (Selleck Chemicals LLC, RRID:SCR_003823)
    Kits for ELISA assays were purchased from R&D Bioscience.
    R&D Bioscience
    suggested: (UMD Bioprocess Scale-Up Facility, RRID:SCR_012703)
    Sequencing data were initially analysed s in the usegalaxy.org platform and then aligned through clustalW, using the Mega 7.0 software.
    clustalW
    suggested: (ClustalW, RRID:SCR_017277)
    The data is available from GEO (https://www.ncbi.nlm.nih.gov/geo/) (39), with accession number GSE92742.
    https://www.ncbi.nlm.nih.gov/geo/
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    Finally, to identify pathways targeted by mefloquine, we applied GSEA (40) using KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways (41).
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    GSEA computes an enrichment score that measures how much a pathway is enriched in differentially expressed genes.
    GSEA
    suggested: (SeqGSEA, RRID:SCR_005724)
    Differentially expressed gene comprising the SARS-CoV-2 infection expression signature were identified using the R package edgeR (43).
    edgeR
    suggested: (edgeR, RRID:SCR_012802)
    Here we used the CMAP pipeline (37,38), comparing the mefloquine expression signature with the SARS-CoV-2 infection expression signature.
    CMAP
    suggested: (CMAP, RRID:SCR_009034)
    The dose-response curves used to calculate EC50 and CC50 values were generated by variable slope plot from Prism GraphPad software 8.0.
    Prism GraphPad
    suggested: None

    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 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.

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  3. Version published to 10.1101/2021.07.21.451321 on bioRxiv