Repurposing screen highlights broad-spectrum coronavirus antivirals and their host targets

  1. Sibylle Haid
  2. Alina Matthaei
  3. Melina Winkler
  4. Svenja M. Sake
  5. Antonia P. Gunesch
  6. Jessica Rückert
  7. Gabrielle Vieyres
  8. David Kühl
  9. Tu-Trinh Nguyen
  10. Lisa Lasswitz
  11. Francisco Zapatero
  12. Graham Brogden
  13. Gisa Gerold
  14. Bettina Wiegmann
  15. Ursula Bilitewski
  16. Mark Brönstrup
  17. Thomas F. Schulz
  18. Thomas Pietschmann

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Abstract

Libraries composed of licensed drugs represent a vast repertoire of molecules modulating physiologic processes in humans, thus providing unique opportunities for discovery of host targeting antivirals. We interrogated the ReFRAME repurposing library with 12,993 molecules for broad-spectrum coronavirus antivirals and discovered 134 compounds inhibiting an alphacoronavirus, mapping to 59 molecular target categories. Dominant targets included the 5-hydroxytryptamine receptor and dopamine receptor and cyclin-dependent kinase inhibitors. Counter-screening with SARS-CoV-2 and validation in primary cells identified Phortress, an aryl hydrocarbon receptor (AHR) ligand, Bardoxolone and Omaveloxolone, two nuclear factor, erythroid 2 like 2 (NFE2L2) activators as inhibitors of both alpha- and betacoronaviruses. The landscape of coronavirus targeting molecules provides important information for the development of broad-spectrum antivirals reinforcing pandemic preparedness.

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  1. ScreenIT

    SciScore for 10.1101/2021.07.14.452343: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: All patients (>18 years of age) gave informed consent for tissue donation and the project was approved by the local ethic committee at MHH (ethic vote 3346/2016).
    IRB: All patients (>18 years of age) gave informed consent for tissue donation and the project was approved by the local ethic committee at MHH (ethic vote 3346/2016).
    Field Sample Permit: Pathway analysis: Pathway analysis and acquisition of metadata about the ReFRAME drug repurposing collection was mainly conducted on the basis of information provided by Scripps Research Institute (https://reframedb.org)
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    BlindingThe analysis pipeline was elaborated and optimised using pictures of cells infected with a blind dilution series of SARS-CoV-2.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: Cells were tested negative for contaminations with mycoplasma (Eurofins). 2.8.

    Table 2: Resources

    Antibodies
    SentencesResources
    Alexa-Fluor Plus 488 anti-mouse antibody (1:1,000; Invitrogen #A32723) and DAPI (1:10,000; Invitrogen #D21490).
    anti-mouse
    suggested: (Thermo Fisher Scientific Cat# A32723, RRID:AB_2633275)
    Experimental Models: Cell Lines
    SentencesResources
    , Vero E6 cells (Chlorocebus aethiops) and Calu-3 cells (Homo sapiens sapiens) (both from Stefan Pöhlmann), Huh-7.5 cells (Homo sapiens sapiens, Charles M.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Huh-7.5
    suggested: RRID:CVCL_7927)
    Rice), MRC-5 cells (Homo sapiens sapiens, Volker Thiel)
    MRC-5
    suggested: ICLC Cat# HL95001, RRID:CVCL_0440)
    , A549 cells (Homo sapiens sapiens, ATCC-CCL-185; Lot 59239596),
    A549
    suggested: None
    2.2. HCoV 229E screening: Huh-7.5/F-Luc cells were seeded in black 384-well plates with a flat clear bottom (Corning #3764) at a density of 3×103 cells/well in phenol-red free Dulbecco’s modified Eagle medium (DMEM; Gibco #31053-028) supplemented with 2 mM L-glutamine (Gibco #25030024), 100 µg/mL streptomycin and 100 U/mL penicillin (Gibco; #15140122), 10% fetal calf serum (FCS, Capricorn Scientific #FBS-11A) and non-essential amino acids (NEAA; Gibco #11140035) one day prior to infection.
    Huh-7.5/F-Luc
    suggested: None
    At 50-80% confluence, Vero cells were inoculated with 500 µL of SARS-CoV-2 (strain SARS-CoV-2/München-1.2/2020/984; p3) in a total of 10 mL of Advanced MEM and incubated at 37°C (5% CO2) under BSL-3 conditions adapted for infectious respiratory viruses.
    Vero
    suggested: None
    Stocks were titrated on Vero and Calu-3 (Finkbeiner et al., 1993) cells (ATCC Cat. #HTB-55 and RRID:CVCL_0609, respectively), cultured in DMEM containing 1% NEAA, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 10% FCS.
    Calu-3
    detected: (ATCC Cat# HTB-55, RRID:CVCL_0609)
    SARS-CoV-2 infection in Calu-3 cells and primary human airway epithelial cells: For infection with SARS-CoV-2, Calu-3 cells (Finkbeiner et al., 1993) were seeded in 96-well plates 48h prior to virus inoculation at a density of 5×104 in DMEM (Gibco #41965039) supplemented as described above.
    Calu-3
    suggested: None
    Software and Algorithms
    SentencesResources
    2.2. HCoV 229E screening: Huh-7.5/F-Luc cells were seeded in black 384-well plates with a flat clear bottom (Corning #3764) at a density of 3×103 cells/well in phenol-red free Dulbecco’s modified Eagle medium (DMEM; Gibco #31053-028) supplemented with 2 mM L-glutamine (Gibco #25030024), 100 µg/mL streptomycin and 100 U/mL penicillin (Gibco; #15140122), 10% fetal calf serum (FCS, Capricorn Scientific #FBS-11A) and non-essential amino acids (NEAA; Gibco #11140035) one day prior to infection.
    Gibco
    suggested: None
    Viral titer (TCID50/mL) were quantified based on the Spearman and Kärber method using a calculator developed by Marco Binder, Heidelberg University.
    Binder
    suggested: (Binder, RRID:SCR_016437)
    Rolling-ball background subtraction was performed for both channels using the ImageJ command (Schneider et al., 2012).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    The percentage of positive cells per condition was grouped in Python 3.8.4 and plotted against the relative cell density (overall cell count per condition relative to the overall cell count in the infected and DMSO-treated control). 2.7.
    Python
    suggested: (IPython, RRID:SCR_001658)
    (Janes et al., 2018) and the Ingenuity Pathway Analysis software suite (IPA, QIAGEN Inc.,
    Ingenuity Pathway Analysis
    suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)
    To complement this information, the following sources were used: DrugBank (https://go.drugbank.com/
    DrugBank
    suggested: (DrugBank, RRID:SCR_002700)
    , PubChem (Kim et al., 2019), Guide to PHARMACOLOGY (Armstrong et al., 2020) and NCBI PubMed (Coordinators, 2018).
    PubChem
    suggested: (PubChem, RRID:SCR_004284)
    PubMed
    suggested: (PubMed, RRID:SCR_004846)
    Data was processed via Microsoft Excel (Version 2016)
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    , GraphPad Prism (Version 8) and Adobe Illustrator.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04494646CompletedBARCONA: A Study of Effects of Bardoxolone Methyl in Partici…
    NCT04069026RecruitingA First-in-Humans Dose Finding Study for an Aryl Hydrocarbon…

    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.
    • Thank you for including a protocol registration statement.

    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 10.1101/2021.07.14.452343v1 on bioRxiv