Arrayed multicycle drug screens identify broadly acting chemical inhibitors for repurposing against SARS-CoV-2

  1. Luca Murer
  2. Romain Volle
  3. Vardan Andriasyan
  4. Nicole Meili
  5. Liliane Yang
  6. Daniela Sequeira
  7. Afonso Gomez-Gonzalez
  8. Anthony Petkidis
  9. Dominik Olszewski
  10. Michael Bauer
  11. Maarit Suomalainen
  12. Fabien Kuttler
  13. Gerardo Turcatti
  14. Urs F. Greber

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Abstract

Coronaviruses (CoVs) circulate in humans and animals, and expand their host range by zoonotic and anthroponotic transmissions. Endemic human CoVs, such as 229E and OC43 cause limited respiratory disease, and elicit short term anti-viral immunity favoring recurrent infections. Yet, severe acute respir-atory syndrome (SARS)-CoV-2 spreads across the globe with unprecedented impact on societies and economics. The world lacks broadly effective and affordable anti-viral agents to fight the pandemic and reduce the death toll. Here, we developed an image-based multicycle replication assay for focus for-mation of α-coronavirus hCoV-229E-eGFP infected cells for screening with a chemical library of 5440 compounds arrayed in 384 well format. The library contained about 39% clinically used compounds, 26% in phase I, II or III clinical trials, and 34% in preclinical development. Hits were counter-selected against toxicity, and challenged with hCoV-OC43 and SARS-CoV-2 in tissue culture and human bronchial and nasal epithelial explant cultures from healthy donors. Fifty three compounds inhibited hCoV-229E-GFP, 39 of which at 50% effective concentrations (EC50) < 2μM, and were at least 2-fold separated from toxicity. Thirty nine of the 53 compounds inhibited the replication of hCoV-OC43, while SARS-CoV-2 was inhibited by 11 compounds in at least two of four tested cell lines. Six of the 11 compounds are FDA-approved, one of which is used in mouth wash formulations, and five are systemic and orally available. Here, we demonstrate that methylene blue (MB) and mycophenolic acid (MPA), two broadly available low cost compounds, strongly inhibited shedding of infectious SARS-CoV-2 at the apical side of the cultures, in either pre- or post-exposure regimens, with somewhat weaker effects on viral RNA release indicated by RT-qPCR measurements. Our study illustrates the power of full cycle screens in repurposing clinical compounds against SARS-CoV-2. Importantly, both MB and MPA reportedly act as immunosuppressants, making them interesting candidates to counteract the cytokine storms affecting COVID-19 patients.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.30.437771: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were permeabilized with 0.2% Triton-X-100 for 5 minutes, washed with PBS, incubated with primary antibodies against coronavirus nucleoprotein (
    coronavirus nucleoprotein
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Huh7-ACE2, HeLa-ACE2 and A549-ACE2 were generated by stable transfection with a lentiviral vector (pLVX-ACE2-IRES-BSD)
    Huh7-ACE2
    suggested: None
    Parental HeLa and A549 cells were obtained from ATCC.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    The parental HeLa cell line additionally expressed an inducible eCas9 and a non-targeting sgRNA.
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    Cell culture: Huh7, Huh7-ACE2, HeLa-ACE2 and A549-ACE2 cells were maintained in Dulbecco’s Modified Eagle Medium (D6429; Sigma-Aldrich, St. Louis, USA) supplemented with 10% fetal bovine serum (FBS, 10270; Invitrogen, Carlsbad, USA)
    Huh7
    suggested: None
    A549-ACE2
    suggested: None
    SARS-CoV2 produced at the apical surface were collected at different time post infection by 20 min apical wash and quantified in parallel by rt-qPCR and TCID50 titration in VeroE6 cells.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Experimental Models: Organisms/Strains
    SentencesResources
    , Merck KGaA, Otava Ltd., Pharmeks Ltd., Ramidus AB, SYN-kinase, Santa Cruz Biotechnology, Selleck Chemicals LLC
    AB
    suggested: RRID:BDSC_203)
    Software and Algorithms
    SentencesResources
    Statistical analyses were performed using GraphPad Prism 8.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Post-processing: Results obtained by image analysis with Plaque2.0 were annotated and filtered using R version 4.0.2 in RStudio Version 1.3.1056.
    RStudio
    suggested: (RStudio, RRID:SCR_000432)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04784559Not yet recruitingTrial to Determine the Efficacy/Safety of Plitidepsin vs Con…
    NCT04629703RecruitingDouble-Blind, Randomized, Placebo-Controlled, Adaptive Desig…

    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.

    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 10.1101/2021.03.30.437771v1 on bioRxiv