Clofoctol inhibits SARS-CoV-2 replication and reduces lung pathology in mice

  1. Sandrine Belouzard
  2. Arnaud Machelart
  3. Valentin Sencio
  4. Thibaut Vausselin
  5. Eik Hoffmann
  6. Nathalie Deboosere
  7. Yves Rouillé
  8. Lowiese Desmarets
  9. Karin Séron
  10. Adeline Danneels
  11. Cyril Robil
  12. Loïc Belloy
  13. Camille Moreau
  14. Catherine Piveteau
  15. Alexandre Biela
  16. Alexandre Vandeputte
  17. Séverine Heumel
  18. Lucie Deruyter
  19. Julie Dumont
  20. Florence Leroux
  21. Ilka Engelmann
  22. Enagnon Kazali Alidjinou
  23. Didier Hober
  24. Priscille Brodin
  25. Terence Beghyn
  26. François Trottein
  27. Benoît Déprez
  28. Jean Dubuisson

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Abstract

Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC 50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.

Summary

Antivirals targeting SARS-CoV-2 are sorely needed. In this study, we screened a library of drug compounds and identified clofoctol as an antiviral against SARS-CoV-2. We further demonstrated that, in vivo, this compound reduces inflammatory gene expression and lowers pulmonary pathology.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2021.06.30.450483: (What is this?)

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

    Table 1: Rigor

    EthicsEuthanasia Agents: For infection, mice (both sexes) were anesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and then intranasally infected with 50 μl of DMEM containing (or not, in a mock sample) 5×102 TCID50 of hCoV-19_IPL_France strain of SARS-CoV-2 (NCBI MW575140).
    IRB: The experimental protocols using animals were approved by the institutional ethical committee “Comité d’Ethique
    Field Sample Permit: The animal study was authorized by the “Education, Research and Innovation Ministry” under registration number APAFIS#25517-2020052608325772v3.
    Sex as a biological variablePharmacokinetic study: Clofoctol diluted in 1.75% final Kolliphor® RH40 (07076, Sigma) and 1.4% final ethanol in a sodium chloride solution (0.9%) was used for intraperitoneal (i.p.) injection (62.5 mg/kg in females and 50 mg/kg in male).
    RandomizationCompounds were spotted in a randomized order on the plates during the primary screen.
    BlindingInvestigators were blinded to allocation during the primary screen and the corresponding validation, during both assay performance and outcome assessment.
    Power AnalysisData reporting: No statistical methods were used to predetermine sample size.
    Cell Line AuthenticationAuthentication: Dose response curves, EC50 calculations and hit validations: The selected hits were further validated in a 6-point dose-response confirmation assay for EC50 determination.

    Table 2: Resources

    Antibodies
    SentencesResources
    Infected cells were detected by using an anti-dsRNA (J2 monoclonal antibody, Scicons) diluted in blocking buffer to detect the presence of replicating SARS-CoV-2 virus as previously determined29.
    anti-dsRNA ( J2
    suggested: None
    After a 30-min incubation, cells were rinsed 3 times for 5 min in PBS and incubated for 30 min with a cyanine 3-conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch) and DAPI (4’,6-diamidino-2-phenylindole).
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    CCL-81), Vero-E6 cells (ATCC, CRL-1586), Huh-7 cells27 and HEK293T/17 cells (ATCC, CRL-11268) were grown at 37°C with 5% CO2 in Dulbecco’s modified eagle medium (DMEM, Gibco) supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Eurobio)
    Vero-E6
    suggested: None
    HEK293T/17
    suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
    One day prior to infection, Vero-81 cells were seeded in 384-well plates, as previously described.
    Vero-81
    suggested: None
    Viral secretion: Vero-81 and Vero-81-TMPRSS2 cells were infected at a MOI of 0.25 for 1h, then the cells were rinsed 3 times with PBS and further incubated in the presence of increasing concentrations of clofoctol for 16h.
    Vero-81-TMPRSS2
    suggested: None
    Briefly, HEK293T cells were co-transfected with a plasmid encoding Gag-Pol (pTG-Gag-Pol), a plasmid encoding the envelope glycoprotein and a plasmid containing a minigenome with a Firefly luciferase reporter gene.
    HEK293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    After 48h of incubation, cell supernatants were collected, filtered and used to transduce Huh-7 cells expressing human ACE2 in the presence of increasing concentrations of clofoctol or CQ.
    Huh-7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    Viability assay: Vero cells, Huh-7 cells or Calu-3 cells were plated in 96-well plates and were then incubated the next day in 100 μl of culture medium containing increasing concentrations of clofoctol for 24h.
    Vero
    suggested: None
    Calu-3
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Experimental infection of K18-hACE2 transgenic mice: Eight week-old K18-human ACE2 expressing C57BL/6 mice (B6.Cg-Tg(K18-hACE2)2Prlmn/J) were purchased from the Jackson Laboratory.
    C57BL/6
    suggested: None
    B6.Cg-Tg(K18-hACE2)2Prlmn/J
    suggested: None
    Recombinant DNA
    SentencesResources
    Lentiviral vectors expressing TMPRSS2 were produced by transfection of HEK293T cells with pTRIP-TMPRSS2, phCMV-VSVG and HIV gag-pol in the presence of Turbofect (Life Technologies) according to the manufacturer’s instruction.
    pTRIP-TMPRSS2
    suggested: None
    Software and Algorithms
    SentencesResources
    Detection was carried out by chemoluminescence (Pierce) and signals were quantified by using the gel quantification function of ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Analysis of the effect of the drug on translation: A plasmid containing a synthetic gene encompassing the 5’-UTR (nucleotides 1-265) and the 3’UTR (nucleotides 29675-29903) of SARS-CoV-2 isolate Wuhan-Hu-1 (Genebank NC_045512.2) separated by two head-to-tail BbsI sites was produced by GeneCust.
    GeneCust
    suggested: None
    Specific primers were designed using Primer Express software (Applied Biosystems, Villebon-sur-Yvette, France) and ordered to Eurofins Scientifics (Ebersberg, Germany).
    Primer Express
    suggested: (Primer Express, RRID:SCR_014326)
    All statistical analysis was performed using GraphPad Prism v6 software.
    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: 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

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.06.30.450483 on bioRxiv