In silico identification and validation of inhibitors of the interaction between neuropilin receptor 1 and SARS-CoV-2 Spike protein

  1. Samantha Perez-Miller
  2. Marcel Patek
  3. Aubin Moutal
  4. Carly R. Cabel
  5. Curtis A. Thorne
  6. Samuel K. Campos
  7. Rajesh Khanna

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Abstract

Neuropilin-1 (NRP-1) is a multifunctional transmembrane receptor for ligands that affect developmental axonal growth and angiogenesis. In addition to a role in cancer, NRP-1 is a reported entry point for several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19). The furin cleavage product of SARS-CoV-2 Spike protein takes advantage of the vascular endothelial growth factor A (VEGF-A) binding site on NRP-1 which accommodates a polybasic stretch ending in a C-terminal arginine. This site has long been a focus of drug discovery efforts for cancer therapeutics. We recently showed that interruption of the VEGF-A/NRP-1 signaling pathway ameliorates neuropathic pain and hypothesize that interference of this pathway by SARS-CoV-2 spike protein interferes with pain signaling. Here, we report hits from a small molecule and natural product screen of nearly 0.5 million compounds targeting the VEGF-A binding site on NRP-1. We identified nine chemical series with lead- or drug-like physico-chemical properties. Using an ELISA, we demonstrate that six compounds disrupt VEGF-A-NRP-1 binding more effectively than EG00229, a known NRP-1 inhibitor. Secondary validation in cells revealed that almost all tested compounds inhibited VEGF-A triggered VEGFR2 phosphorylation. Two compounds displayed robust inhibition of a recombinant vesicular stomatitis virus protein that utilizes the SARS-CoV-2 Spike for entry and fusion. These compounds represent a first step in a renewed effort to develop small molecule inhibitors of the VEGF-A/NRP-1 signaling for the treatment of neuropathic pain and cancer with the added potential of inhibiting SARS-CoV-2 virus entry.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.22.308783: (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
    Anti-VEGFR2 pY1175 was used to detect the activation of the pathway triggered by VEGF-A165 in the cells.
    Anti-VEGFR2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Titer was determined to be approximately 1×107 PFU/mL as determined by median tissue culture infectious dose (TCID50) assay on Vero-E6-TMPRSS2 cells.
    Vero-E6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Screening compounds for VSV-eGFP-SARS-CoV-2 inhibition: African green monkey kidney (Vero)-E6-TMPRSS2 cells were plated at 15,000 cells per well in black/clear bottom 96 well tissue culture plates (Thermo Fisher Scientific 165305).
    Vero)-E6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    These were prepared for screening in LigPrep using the OPLS3e force field, neutral ionization, desalting, and tautomer generation.
    LigPrep
    suggested: (Ligprep, RRID:SCR_016746)
    Sum GFP fluorescence intensity, normalized to cell count by HCS CellMask Blue (Thermo #H32720), was measured and for each well and plotted with Prism 6 (GraphPad Software).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    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

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  2. Version published to 10.1101/2020.09.22.308783v1 on bioRxiv