Enisamium is a small molecule inhibitor of the influenza A virus and SARS-CoV-2 RNA polymerases

  1. Alexander P Walker
  2. Haitian Fan
  3. Jeremy R Keown
  4. Victor Margitich
  5. Jonathan M Grimes
  6. Ervin Fodor
  7. Aartjan J W te Velthuis

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Abstract

Influenza A virus and coronavirus strains cause a mild to severe respiratory disease that can result in death. Although vaccines exist against circulating influenza A viruses, such vaccines are ineffective against emerging pandemic influenza A viruses. Currently, no vaccine exists against coronavirus infections, including pandemic SARS-CoV-2, the causative agent of the Coronavirus Disease 2019 (COVID-19). To combat these RNA virus infections, alternative antiviral strategies are needed. A key drug target is the viral RNA polymerase, which is responsible for viral RNA synthesis. In January 2020, the World Health Organisation identified enisamium as a candidate therapeutic against SARS-CoV-2. Enisamium is an isonicotinic acid derivative that is an inhibitor of multiple influenza B and A virus strains in cell culture and clinically approved in 11 countries. Here we show using in vitro assays that enisamium and its putative metabolite, VR17-04, inhibit the activity of the influenza virus and the SARS-CoV-2 RNA polymerase. VR17-04 displays similar efficacy against the SARS-CoV-2 RNA polymerase as the nucleotide analogue remdesivir triphosphate. These results suggest that enisamium is a broad-spectrum small molecule inhibitor of RNA virus RNA synthesis, and implicate it as a possible therapeutic option for treating SARS-CoV-2 infection. Unlike remdesivir, enisamium does not require intravenous administration which may be advantageous for the development of COVID-19 treatments outside a hospital setting.

Importance

Influenza A virus and SARS-CoV-2 are respiratory viruses capable of causing pandemics, and the latter is responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Both viruses encode RNA polymerases which transcribe their RNA genomes and are important targets for antiviral drugs including remdesivir. Here, we show that the antiviral drug enisamium inhibits the RNA polymerases of both influenza A virus and SARS-CoV-2. Furthermore, we show that a putative metabolite of enisamium is a more potent inhibitor, inhibiting the SARS-CoV-2 RNA polymerase with similar efficiency to remdesivir. Our data offer insight into the mechanism of action for enisamium, and implicate it as a broad-spectrum antiviral which could be used in the treatment of SARS-CoV-2 infection.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.21.053017: (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
    Western analysis was performed on 8% SDS-PAGE and PA antibody GTX125932 (GeneTex) FluPol in vitro activity assays: The subunits of the A/WSN/33 (H1N1) influenza virus FluPol with a Tandem Affinity Purification (TAP)-tag at the C-terminus of PB2 (pcDNA3-PB1, pcDNA3-PB2-TAP, pcDNA3-PA) were expressed in HEK 293T cells and purified as heterotrimeric complex using IgG sepharose chromatography as described previously(29).
    PA
    suggested: (GeneTex Cat# GTX125932, RRID:AB_11175740)
    H1N1
    suggested: None
    pcDNA3-PB1 , pcDNA3-PB2-TAP , pcDNA3-PA
    suggested: None
    pcDNA3-PB2-TAP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Plaque assays were performed on MDCK cells in MEM containing 0.5% FCS with a 1% agarose overlay and grown for 2 days at 37 °C.
    MDCK
    suggested: None
    Western analysis was performed on 8% SDS-PAGE and PA antibody GTX125932 (GeneTex) FluPol in vitro activity assays: The subunits of the A/WSN/33 (H1N1) influenza virus FluPol with a Tandem Affinity Purification (TAP)-tag at the C-terminus of PB2 (pcDNA3-PB1, pcDNA3-PB2-TAP, pcDNA3-PA) were expressed in HEK 293T cells and purified as heterotrimeric complex using IgG sepharose chromatography as described previously(29).
    HEK 293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    FAV00A-treated A549 cell extracts: Approximately 106 A549 cells treated with FAV00A in DMEM containing 10% FCS were lysed through sonication in 40 μl buffer A (10 mM HEPES-NaOH, pH 7.5, 0.1% NP-40, 150 mM NaCl, 5% glycerol, and 1 mM DTT) for 10 seconds.
    A549
    suggested: None
    Software and Algorithms
    SentencesResources
    Data were analysed using ImageJ and Prism 8 (GraphPad).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: 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.

    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 10.1101/2020.04.21.053017v2 on bioRxiv
  3. Version published to 10.1101/2020.04.21.053017v1 on bioRxiv