N4-hydroxycytidine and inhibitors of dihydroorotate dehydrogenase synergistically suppress SARS-CoV-2 replication

  1. Kim M. Stegmann
  2. Antje Dickmanns
  3. Natalie Heinen
  4. Uwe Groß
  5. Dirk Görlich
  6. Stephanie Pfaender
  7. Matthias Dobbelstein

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Abstract

Effective therapeutics to inhibit the replication of SARS-CoV-2 in infected individuals are still under development. The nucleoside analogue N4-hydroxycytidine (NHC), also known as EIDD-1931, interferes with SARS-CoV-2 replication in cell culture. It is the active metabolite of the prodrug Molnupiravir (MK-4482), which is currently being evaluated for the treatment of COVID-19 in advanced clinical studies. Meanwhile, inhibitors of dihydroorotate dehydrogenase (DHODH), by reducing the cellular synthesis of pyrimidines, counteract virus replication and are also being clinically evaluated for COVID-19 therapy. Here we show that the combination of NHC and DHODH inhibitors such as teriflunomide, IMU-838/vidofludimus, and BAY2402234, strongly synergizes to inhibit SARS-CoV-2 replication. While single drug treatment only mildly impaired virus replication, combination treatments reduced virus yields by at least two orders of magnitude. We determined this by RT-PCR, TCID 50 , immunoblot and immunofluorescence assays in Vero E6 and Calu-3 cells infected with wildtype and the Alpha and Beta variants of SARS-CoV-2. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC in nascent viral RNA, thus precluding the correct synthesis of the viral genome in subsequent rounds of replication, thereby inhibiting the production of replication competent virus particles. This concept was further supported by the rescue of replicating virus after addition of pyrimidine nucleosides to the media. Based on our results, we suggest combining these drug candidates, which are currently both tested in clinical studies, to counteract the replication of SARS-CoV-2, the progression of COVID-19, and the transmission of the disease within the population.

SIGNIFICANCE

  • The strong synergy displayed by DHODH inhibitors and the active compound of Molnupiravir might enable lower concentrations of each drug to antagonize virus replication, with less toxicity.

  • Both Molnupiravir and DHODH inhibitors are currently being tested in advanced clinical trials or are FDA-approved for different purposes, raising the perspective of rapidly testing their combinatory efficacy in clinical studies.

  • Molnupiravir is currently a promising candidate for treating early stages of COVID-19, under phase II/III clinical evaluation. However, like Remdesivir, it appears only moderately useful in treating severe COVID-19. Since the combination inhibits virus replication far more strongly, and since DHODH inhibitors may also suppress excessive immune responses, the combined clinical application bears the potential of alleviating the disease burden even at later stages.

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  1. ScreenIT

    SciScore for 10.1101/2021.06.28.450163: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: Cells were authenticated by the Leibniz-Institute DSMZ and routinely tested and ensured to be negative for mycoplasma contamination

    Table 2: Resources

    Antibodies
    SentencesResources
    The secondary Alexa Fluor 546 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-mouse IgG (Invitrogen, 1:500, diluted in blocking solution) antibodies were added together with 4′,6-diamidino-2-phenylindole (DAPI) for 1.5 hours at room temperature.
    anti-rabbit IgG
    suggested: None
    To determine the presence of viral proteins, the separated proteins were transferred to a nitrocellulose membrane, blocked in 5% (w/v) non-fat milk in TBS containing 0.1% Tween-20 for 1 hour, and incubated with primary antibodies at 4 °C overnight, followed by incubation with peroxidase-conjugated secondary antibodies (donkey anti-rabbit or donkey anti-mouse IgG, Jackson Immunoresearch).
    anti-rabbit
    suggested: None
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Experimental Model And Methods: Cell culture: Vero E6 cells (Vero C1008) were maintained in Dulbecco’s modified Eagle’s medium (DMEM with GlutaMAX™, Gibco) supplemented with 10% fetal bovine serum (Merck), 50 units/ml penicillin, 50 μg/ml streptomycin (Gibco), 2 µg/ml tetracycline (Sigma) and 10 µg/ml ciprofloxacin (Bayer) at 37°C in a humidified atmosphere with 5% CO2.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Calu-3 cells were maintained in Eagle’s Minimum Essential Medium (EMEM, ATCC) supplemented with 10% fetal bovine serum and penicillin/streptomycin.
    Calu-3
    suggested: None
    Software and Algorithms
    SentencesResources
    Percent cytotoxicity reflects the proportion of LDH released to the media compared to the overall amount of LDH in the cells, and was calculated using the following formula:

    Quantification and statistical analysis: Statistical testing was performed using Graph Pad Prism 6 (RRID:SCR_002798).

    Graph Pad Prism
    detected: 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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04575584Active, not recruitingEfficacy and Safety of Molnupiravir (MK-4482) in Hospitalize…
    NCT04575597RecruitingEfficacy and Safety of Molnupiravir (MK-4482) in Non-Hospita…
    NCT04405739RecruitingThe Safety of Molnupiravir (EIDD-2801) and Its Effect on Vir…
    NCT04379271CompletedA Study to Evaluate the Efficacy, Safety and Tolerability of…

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.06.28.450163v1 on bioRxiv