The natural stilbenoid (–)-hopeaphenol inhibits cellular entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7 and B.1.351 variants

  1. Ian Tietjen
  2. Joel Cassel
  3. Emery T. Register
  4. Xiang Yang Zhou
  5. Troy E. Messick
  6. Frederick Keeney
  7. Lily D. Lu
  8. Karren D. Beattie
  9. Topul Rali
  10. Hildegund C. J. Ertl
  11. Joseph M. Salvino
  12. Rohan A. Davis
  13. Luis J. Montaner

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Abstract

Antivirals are urgently needed to combat the global SARS-CoV-2/COVID-19 pandemic, supplement existing vaccine efforts, and target emerging SARS-CoV-2 variants of concern. Small molecules that interfere with binding of the viral spike receptor binding domain (RBD) to the host ACE2 receptor may be effective inhibitors of SARS-CoV-2 cell entry. Here we screened 512 pure compounds derived from natural products using a high-throughput RBD/ACE2 binding assay and identified (–)-hopeaphenol, a resveratrol tetramer, in addition to vatalbinoside A and vaticanol B, as potent and selective inhibitors of RBD/ACE2 binding and viral entry. For example, (–)-hopeaphenol disrupted RBD/ACE2 binding with a 50% inhibitory concentration (IC50) of 0.11 μM in contrast to an IC50 of 28.3 μM against the unrelated host ligand/receptor binding pair PD-1/PD-L1 (selectivity index = 257.3). When assessed against the USA-WA1/2020 variant, (–)-hopeaphenol also inhibited entry of a VSVΔG-GFP reporter pseudovirus expressing SARS-CoV-2 spike into ACE2-expressing Vero-E6 cells and in vitro replication of infectious virus in cytopathic effect assays (IC50 = 10.2 μM) without cytotoxicity. Notably, (–)- hopeaphenol also inhibited two emerging variants of concern originating from the United Kingdom (B.1.1.7) and South Africa (B.1.351) in both cytopathic effect and spike-containing pseudovirus assays with similar (B.1.1.7) or improved (B.1.351) efficacies over the USA- WA1/2020 variant. These results identify (–)-hopeaphenol and related stilbenoid analogues as potent and selective inhibitors of viral entry across multiple SARS-CoV-2 variants including those with increased infectivity and/or reduced susceptibility to existing vaccines.

Importance

SARS-CoV-2 antivirals are needed to supplement existing vaccine efforts and target emerging viral variants with increased infectivity or reduced susceptibility to existing vaccines. Here we show that (–)-hopeaphenol, a naturally-occurring stilbenoid compound, in addition to its analogues vatalbinoside A and vaticanol B, inhibit SARS-CoV-2 by blocking the interaction of the viral spike protein with the cellular ACE2 entry receptor. Importantly, in addition to inhibiting the early USA-WA1/2020 SARS-CoV-2 variant, hopeaphenol also inhibits emerging variants of concern including B.1.1.7 (“United Kingdom variant”) and B.1.351 (“South Africa variant”), with improved efficacy against B.1.351. (–)-Hopeaphenol therefore represents a new antiviral lead against infection from multiple SARS-CoV-2 variants.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.29.442010: (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.
    BlindingCells were incubated for an additional 4 days, at which point all wells were scored for the presence of CPE by a user blinded to the identity of the wells.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Pseudoviruses were generated in BHK-21/WI-2 cells using a pseudotyped ΔG-GFP (G*ΔG-GFP) rVSV (Kerafast, Boston, MA, USA) in addition to spike sequences cloned into the paT7- Spike plasmid as described previously (21).
    BHK-21/WI-2
    suggested: RRID:CVCL_HB78)
    Viral CPE assays: Vero-E6 cells were plated in D10+ to 20,000 cells per well in 96-well format and incubated for 24 hours.
    Vero-E6
    suggested: None
    Recombinant DNA
    SentencesResources
    Generation of Mpro protein: The codon-optimized gene for SARS-CoV-2 Mpro (or 3CLpro) (GenBank: QHD43415.1 aa 3264-3567) from strain BetaCoV/Wuhan/WIV04/2019 was ordered from IDT (Coralville, IA, USA) and cloned into a HIS-SUMO expression vector (a modified pET-DUET; Novagen, Madison, WI, USA).
    pET-DUET
    suggested: RRID:Addgene_111354)
    Software and Algorithms
    SentencesResources
    Data analysis: For all studies, 50% effective concentrations were calculated using nonlinear regression of a one-side binding model using GraphPad Prism v. 8.4.3 (GraphPad, San Diego, CA, USA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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.

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

    About SciScore

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  2. Version published to 10.1101/2021.04.29.442010 on bioRxiv