Antiviral activity of Molnupiravir precursor NHC against SARS-CoV-2 Variants of Concern (VOCs) and implications for the therapeutic window and resistance

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Abstract

Several regulatory agencies have either licensed or given emergency use approval for treatment of patients at risk of developing severe COVID-19 with the anti-viral drug, Molnupiravir. Recent trials involving Molnupiravir suggested the drug was not as efficacious as earlier studies suggested. This study aimed to: (i) determine the effectiveness of the Molnupiravir active metabolite (NHC) against different SARS-CoV-2 Variants of Concern (VoCs), (ii) establish the therapeutic window of NHC in a human lung cell model, and (iii) and evaluate the genetic barrier to resistance. Dose response assays were performed in parallel to determine the IC50 (the concentration required to inhibit virus titre by 50%) of NHC against different variants. Human ACE-2 A549 cells were treated with NHC at different time points either before, during or after infection with SARS-CoV-2. Multiple passaging in the presence or absence of drug was used to evaluate whether resistance occurred. To obtain genomic information, virus was sequenced at regular intervals. After 20 passages in the presence of the drug, dose response assays and sequencing showed the virus did not appear to have developed resistance. The drug had equivalent activity against four VOCs ranging from 0.04 to 0.16μM IC50. The efficacy of the drug diminished when applied after 24 hours post-infection. Our results suggest that earlier administration in patients, perhaps pre- or post-exposure rather than symptom onset, would be a more effective treatment option.

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  1. SciScore for 10.1101/2021.11.23.469695: (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 Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    ) Human ACE2-A549 cells were grown to confluency and infected at an MOI of 0.1 in either DMEM with 2%FBS and 0.05mg/ml gentamicin, or in the same media containing 0.01µM, 0.1µM, 1µM or 10µM NHC by allowing virus to adsorb to cells in a volume of 100µl for one hour at 37°C, and then topping up to 500µl with the relevant media afterwards.
    ACE2-A549
    suggested: None
    Results In order to find the appropriate concentration range of NHC in hACE2-A549 cells, to investigate the effect on viral biology, Cell-titer Glo Assay (Promega) were used to measure % ATP production in cells.
    hACE2-A549
    suggested: None
    Software and Algorithms
    SentencesResources
    Primer-trimmed bam files were further analysed using DiversiTools (http://josephhughes.github.io/DiversiTools/) with the “-orfs” function to generate the ratio of amino acid change in the reads and coverage at each site of protein in comparison to the reference SARS-CoV-2 genome (MN908947.3).
    DiversiTools
    suggested: None
    The absolute IC50 values were calculated using GraphPad Prism 9, using a non-linear 4-parameter logistic regression in a dose-response curve.
    GraphPad Prism
    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: We detected the following sentences addressing limitations in the study:
    However, in-vitro systems have several limitations in comparison to live models of infection, so results should be interpreted with care, although the mechanism of action will be intra-cellular. Use of molnupiravir in a Syrian hamster model infected with SARS-CoV-2 resulted in a drop in viral load and reduced lung pathology compared to controls Treatment 12 hours post-infection resulted in a protective effect 12 but not at 24 hours post- infection. Further work is required to delineate the true treatment window of the drug in humans with mild to moderate disease. Anti-viral drugs are best given early in infection to reduce viral load. In-vivo ferret models have shown that severity is linked to the size of the viral inoculum18. Therefore, molnupiravir likely acts to reduce disease by reducing viral load in patients, and potentially subsequently reducing transmission. 10,12. One of the main benefits of molnupiravir as opposed to remdesivir is that it can be administered orally. However, as was seen with the Influenza anti-viral, Tamiflu, resistance to anti-virals can develop rapidly develop resistance is necessary, though it is likely that any adaptation for resistance will correspond with a reduction in fitness as seen with remdesivir 20. Use of molnupiravir would likely be most beneficial if used in combination with another treatment, preferably targeting a different part of the viral life cycle as has been used with success for HIV treatment. Finally, molnupiravir has broad ...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04746183RecruitingAGILE (Early Phase Platform Trial for COVID-19)


    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.

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


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