Novel and potent inhibitors targeting DHODH are broad-spectrum antivirals against RNA viruses including newly-emerged coronavirus SARS-CoV-2

  1. Rui Xiong
  2. Leike Zhang
  3. Shiliang Li
  4. Yuan Sun
  5. Minyi Ding
  6. Yong Wang
  7. Yongliang Zhao
  8. Yan Wu
  9. Weijuan Shang
  10. Xiaming Jiang
  11. Jiwei Shan
  12. Zihao Shen
  13. Yi Tong
  14. Liuxin Xu
  15. Yu Chen
  16. Yingle Liu
  17. Gang Zou
  18. Dimitri Lavillete
  19. Zhenjiang Zhao
  20. Rui Wang
  21. Lili Zhu
  22. Gengfu Xiao
  23. Ke Lan
  24. Honglin Li
  25. Ke Xu

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Abstract

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC 50 of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.

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  1. Version published to 10.1007/s13238-020-00768-w
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    SciScore for 10.1101/2020.03.11.983056: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementAll animal experiments were approved by the Institutional Animal Care and Use Committee of Institut Pasteur of Shanghai and Wuhan University.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variableMice infection and drug treatment BALB/c female mice, 6-8 weeks old, were purchased from LingChang Company and bred in the specific pathogen-free animal facility.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Thereafter, singlecell sorting was performed in a 96-well plate to grow single clones, and western blot using the anti-DHODH antibody (Santa Cruz) was performed to detect the expression level of DHODH.
    anti-DHODH
    suggested: None
    The cells were then incubated with the primary antibody (a polyclonal antibody against the NP of a bat SARS-related CoV) after blocking, followed by incubation with the secondary antibody (Alexa 488-labeled goat anti-rabbit, Abcam).
    anti-rabbit, Abcam).
    suggested: None
    Immunostaining was performed using an anti-NP polyclonal antibody conjugated with HRP (Antibody Research Center, Shanghai Institute of Biological Science).
    anti-NP
    suggested: None
    Cells were fixed and permeabilized for staining with anti-viral NP antibody, followed by staining with Alexa 488-labeled secondary antibody.
    anti-viral NP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Materials & Methods Cell lines, virus, and drugs MDCK, A549, Vero E6, Huh7 cells were obtained from the American Type Culture Collection (ATCC).
    Huh7
    suggested: CLS Cat# 300156/p7178_HuH7, CVCL_0336
    BSR T7/5 cells stably expressing the T7 RNA polymerase gene were kindly provided by Gang Zou (Institute Pasteur of Shanghai, China).
    BSR T7/5
    suggested: CCLV Cat# CCLV-RIE 0583, CVCL_RW96
    MDCK, A549, Vero E6, Huh7, 293FT cells were cultured in DMEM (Gibco) supplemented with 10%FBS (Gibco) and 1% P/S (Gibco).
    MDCK
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>293FT</b></div>
        <div>suggested: ATCC Cat# PTA-5077, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_6911">CVCL_6911</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Influenza mini-replicon system and DHODH substrates assay The 293T cells were seeded into 24-well plates at 1×105 cells per well and were transfected with influenza A/WSN/33 virus PB1-, PB2-, PA- and NP- expressing plasmids (100 ng each), the influenza virus-specific RNA polymerase I driven firefly luciferase reporter (pPoⅡ-NP-luc) (100 ng, provide by Han-D Klenk, Marburg University), and the Renilla luciferase reporter pRLSV40 10 ng, Promega).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">BSR-T7/5 cells were transfected with the EBOV mini-genome replication system (NP, VP35, VP30, MG, and L) in the presence of increasing concentrations of Teriflunomide, Brequinar, S312 and S416 respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>BSR-T7/5</b></div>
        <div>suggested: CCLV Cat# CCLV-RIE 0583, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_RW96">CVCL_RW96</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Aliquots of Vero E6 cells were seeded in 96-well plates and then infected with Beta CoV/Wuhan/WIV04/2019 at MOI of 0.03.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero E6</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A549 (WT or DHODH-/-) cells were infected with WSN virus with MOI of 0.01.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>A549</b></div>
        <div>suggested: NCI-DTP Cat# A549, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0023">CVCL_0023</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were processed with Graphpad prism software to calculate EC50 and CC50 values of the compounds.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Graphpad prism</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data availability The protein structure data has been uploaded to the Protein Data Bank with accession number 6M2B. Acknowledgments: This work was supported in part by the National Key Research and Development Program (grant 2018FYA0900801, 2018ZX10101004003001, 2016YFA0502304), the National Natural Science Foundation (NSFC) of China (grants 31922004, 81825020, 81772202), the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” China (grant 2018ZX09711002), Application & Frontier Research Program of Wuhan Government (2019020701011463).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Frontier Research Program</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr></table>

    Results from Barzooka: We also 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 OddPub: Thank you for sharing your data.

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  3. Version published to 10.1101/2020.03.11.983056v1 on bioRxiv