Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase

  1. Hayden T. Pacl
  2. Jennifer L. Tipper
  3. Ritesh R. Sevalkar
  4. Andrew Crouse
  5. Camerron Crowder
  6. UAB Precision Medicine Institute
  7. Shama Ahmad
  8. Aftab Ahmad
  9. Gillian D. Holder
  10. Charles J. Kuhlman
  11. Krishna C. Chinta
  12. Sajid Nadeem
  13. Todd J. Green
  14. Chad M. Petit
  15. Adrie J.C. Steyn
  16. Matthew Might
  17. Kevin S. Harrod

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Abstract

The recent emergence of a novel coronavirus, SARS-CoV-2, has led to the global pandemic of the severe disease COVID-19 in humans. While efforts to quickly identify effective antiviral therapies have focused largely on repurposing existing drugs 1–4 , the current standard of care, remdesivir, remains the only authorized antiviral intervention of COVID-19 and provides only modest clinical benefits 5 . Here we show that water-soluble derivatives of α-tocopherol have potent antiviral activity and synergize with remdesivir as inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Through an artificial-intelligence-driven in silico screen and in vitro viral inhibition assay, we identified D-α-tocopherol polyethylene glycol succinate (TPGS) as an effective antiviral against SARS-CoV-2 and β-coronaviruses more broadly that also displays strong synergy with remdesivir. We subsequently determined that TPGS and other water-soluble derivatives of α-tocopherol inhibit the transcriptional activity of purified SARS-CoV-2 RdRp and identified affinity binding sites for these compounds within a conserved, hydrophobic interface between SARS-CoV-2 nonstructural protein 7 and nonstructural protein 8 that is functionally implicated in the assembly of the SARS-CoV-2 RdRp 6 . In summary, we conclude that solubilizing modifications to α-tocopherol allow it to interact with the SARS-CoV-2 RdRp, making it an effective antiviral molecule alone and even more so in combination with remdesivir. These findings are significant given that many tocopherol derivatives, including TPGS, are considered safe for humans, orally bioavailable, and dramatically enhance the activity of the only approved antiviral for SARS-CoV-2 infection 7–9 .

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  1. Version published to 10.1101/2021.07.13.449251v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.07.13.449251: (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

    Antibodies
    SentencesResources
    Cells were then directly incubated with a rabbit polyclonal antibody against SARS-CoV-2 spike/RBD protein (SinoBiological # 40592-T62), diluted 1:2500 in milk on a rocking platform for 1 hour at room temperature.
    SARS-CoV-2 spike/RBD protein
    suggested: None
    Following this incubation, cells were washed five times with PBS and then incubated with horseradish-peroxidase-conjugated goat-anti-rabbit secondary antibody (Invitrogen # A16104) diluted 1:1000 in milk shaking for 1 hour.
    goat-anti-rabbit
    suggested: None
    For OC43, an identical procedure was followed, except that the primary antibody for viral detection was a monoclonal antibody against the coronavirus group antigen (Milipore Sigma #MAB9013) and the secondary was a horseradish-peroxidase-conjugated goat-anti-mouse antibody (enQuire BioReagents # Q2AB1).
    antibody
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: Stocks of low-passage VeroE6 (ATCC # C1008) and Calu3 (ATCC # HTB-55) cells were stored in liquid nitrogen.
    Calu3
    suggested: None
    Infections were performed for one hour in SF-MEM at 35°C at a multiplicity of infection (MOI) of 0.1 for SARS-CoV-2 in VeroE6 cells, an MOI of 1 for SARS-CoV-2 in Calu3 cells, and an MOI of 0.1 for OC43 in VeroE6 cells.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    Its knowledge graph database incorporates over 180 distinct sources of biomedical knowledge, including the scientific literature (Pubmed), data on approved and unapproved drugs (via FDA and drugbank), and genes (NCBI, UniProt).
    Pubmed
    suggested: (PubMed, RRID:SCR_004846)
    For OC43, an identical procedure was followed, except that the primary antibody for viral detection was a monoclonal antibody against the coronavirus group antigen (Milipore Sigma #MAB9013) and the secondary was a horseradish-peroxidase-conjugated goat-anti-mouse antibody (enQuire BioReagents # Q2AB1).
    BioReagents
    suggested: (Fisher BioReagents, RRID:SCR_003374)
    Whole-well images were then processed in FIJI software48 where they underwent automated well-detection by thresholding and particle analysis followed by gaussian filtering, background flattening, and thresholding of the original image to determine the proportion of the well stained for viral protein.
    FIJI
    suggested: (Fiji, RRID:SCR_002285)
    Acquired images were processed in Gen5 software by background flattening and blurring prior to nuclear segmentation based on Hoescht 33342 staining.
    Gen5
    suggested: (Gen5, RRID:SCR_017317)
    TrimGalore! (version 0.6.6) (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) was used to trim the raw sequence FASTQ reads of primer adapter contamination (parameters used: --paired --trim-n --trim1 --nextseq 20).
    https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/
    suggested: (Trim Galore, RRID:SCR_011847)
    STAR (version 2.7.7a) was used to align the trimmed FASTQ reads to the combined Vero and SARS-CoV-2 Washington strain genomes (parameters used: --outReadsUnmapped Fastx --outSAMtype BAM SortedByCoordinate --outSAMattributes All)59.
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Following alignment, HTSeq-count (version 0.13.5) was used to count the number of reads mapping to each gene (parameters used: -m union -r pos -t exon -i gene_id -a 10 -s no -f bam)60.
    HTSeq-count
    suggested: (htseq-count, RRID:SCR_011867)
    The ten lowest energy solutions were then analyzed, and figures were generated in Pymol using a combination of the pose with the lowest energy as well as all poses found.
    Pymol
    suggested: (PyMOL, RRID:SCR_000305)
    Free energy of binding calculations are performed using the intermolecular component of the lowest-scoring conformation as calculated via AutoDock Vina’s scoring function61
    AutoDock
    suggested: (AutoDock, RRID:SCR_012746)

    Results from OddPub: Thank you for sharing your code.

    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 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 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.

    About SciScore

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  3. Version published to 10.1101/2021.07.13.449251v1 on bioRxiv