Targeting an evolutionarily conserved “E-L-L” motif in spike protein to identify a small molecule fusion inhibitor against SARS-CoV-2

  1. Indrani Das Jana
  2. Prabuddha Bhattacharya
  3. Karthick Mayilsamy
  4. Saptarshi Banerjee
  5. Gourab Bhattacharje
  6. Sayan Das
  7. Seemanti Aditya
  8. Anandita Ghosh
  9. Andrew R McGill
  10. Syamanthak Srikrishnan
  11. Amit Kumar Das
  12. Amit Basak
  13. Shyam S Mohapatra
  14. Bala Chandran
  15. Devesh Bhimsaria
  16. Subhra Mohapatra
  17. Arunava Roy
  18. Arindam Mondal

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Abstract

As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key toward sustenance. Here, we identify an evolutionarily conserved “Ex3Lx6L” (“E-L-L”) motif present within the HR2 domain of all human and nonhuman coronavirus spike (S) proteins that play a crucial role in stabilizing its postfusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduce the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this “E-L-L” motif and impedes the formation of 6-HB, thus effectively inhibiting SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy in blocking S protein-mediated viral entry, mutations within the “E-L-L” motif rendered the protein completely resistant to the drug, establishing its specificity toward this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective toward all major variants of concerns of SARS-CoV-2, including Beta, Kappa, Delta, and Omicron. Together, we show that this conserved essential “E-L-L” motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.

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  1. Version published to 10.1093/pnasnexus/pgac198
  2. Version published to 10.1101/2022.03.16.484554v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2022.03.16.484554: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies and Drugs: Goat polyclonal antibody against human ACE2 (AF933) and rabbit polyclonal antibody against SARS CoV-2 Spike protein (NB100-56578) were obtained from Novus Biologicals (
    human ACE2
    suggested: None
    antibody against SARS CoV-2 Spike protein
    suggested: None
    Cells were then washed two times with PBS and incubated with an anti-spike antibody diluted (1:200) in PBS for 1 h at 37°C.
    anti-spike
    suggested: None
    Proteins were transferred to PVDF membrane, probed using anti SARS CoV-2 S antibody (Novus Biologicals, USA) and visualized with Chemiluminescent Reagent (Pierce).
    anti SARS CoV-2 S
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: Human embryonic kidney (HEK) 293T cells (ATCC #CRL-3216), Madin Darby Canine Kidney (MDCK) cells (ATCC #CCL-34), and African green monkey kidney epithelial (Vero) cells (ATCC #CCL-81) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) along with penicillin and streptomycin antibiotics (Gibco) at 37°C and 5% CO2.
    HEK
    suggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)
    293T
    suggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)
    All SARS-CoV-2 stocks were produced and purified from supernatants of Vero-E6 ACE2 cells, infected with different variants of SARS-CoV-2 or SARS-CoV-2-mNG virus, at MOI of 0.1 in 10 ml MEM supplemented with 5% FBS. 72 hpi, virus-containing supernatants were harvested, centrifuged (500g, 10 min), filtered (0.45 μm), mixed with 10% SPG buffer (ATCC #MD9692), aliquoted and stored at −80°C.
    Vero-E6 ACE2
    suggested: None
    For quantification of virus titers, viral stocks were serially diluted (10-fold) in serum-free medium and inoculated on 1×105 Vero E6-ACE2 cells, followed by determination of plaques forming units (PFU) after 72 h of infection on confluent Vero-E6-ACE2 cells.
    Vero E6-ACE2
    suggested: None
    Vero-E6-ACE2
    suggested: None
    Herpes Simplex Virus 1 (HSV-1 KOS) was propagated and purified from Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    SARS-CoV-2 S and VSVG pseudotyped lentivirus generation: Lentivirus virus particles pseudotyped with full-length WT and mutant SARS-CoV-2 S protein were generated by transfecting HEK293T cells as described by Katharine et. al (Crawford et al., 2020).
    HEK293T
    suggested: None
    Briefly, the influenza A reporter virus was preincubated with Posaconazole for 30 min at RT and subsequently used to infect MDCK cells seeded in a 96-well plate.
    MDCK
    suggested: None
    For evaluation of the effect of posaconazole on the surface localization of the spike protein, Caco-2 cells were pre-treated with the drug or the vehicle control for 12 h and then infected with SARS-CoV-2 in the presence of the drug.
    Caco-2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Plasmids: Plasmid HDM-IDTSpike-fixK, expressing a codon-optimized spike protein from SARS-CoV-2, Wuhan-Hu-1 strain (Genbank NC_045512) under a CMV promoter (BEI catalog number NR-52514), was used in this study.
    Wuhan-Hu-1
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmid pHAGE2-EF1aInt-ACE2-WT, expressing the human ACE2 gene (BEI #NR-52514) was used for all ACE2 overexpression studies.
    pHAGE2-EF1aInt-ACE2-WT
    suggested: None
    Plasmids pCMV VSV-G, expressing the VSV-G (addgene#138479) (Gee et al., n.d.), pcDNA3.3-SARS2-B.1.617.2 SARS-CoV-2, expressing the spike protein of the Delta variant (addgene#172320), pcDNA3.3-SARS2-B.1.617.1, expressing the spike protein of the Kappa variant (addgene#172319)(Cho et al., 2021), and SARS-CoV-2 Omicron Strain S gene Human codon_pcDNA3.1(+) expressing the spike protein of the Omicron variant (GenScript# MC_0101274) were used as indicated.
    pCMV VSV-G
    suggested: None
    pcDNA3.3-SARS2-B.1.617.2 SARS-CoV-2
    suggested: None
    pcDNA3.3-SARS2-B.1.617.1
    suggested: RRID:Addgene_172319)
    Briefly, the lentiviral backbone plasmid pHAGE-CMV-Luc2-IRES-ZsGreen-W (BEI #NR-52516) that uses a CMV promoter to express luciferase followed by an IRES and ZsGreen, was co-transfected with plasmids pHDM-Hgpm2, expressing the HIV-1 gag and pol (BEI #NR-52517), pRC-CMV-rev1b, expressing the HIV-1 rev (BEI #NR-52519), pHDM-tat1b, expressing the HIV-1 Tat (BEI #NR NR-52518), and HDM-IDTSpike-fixK or its mutants at a 4.6:1:1:1:1.6 ratio.
    pHAGE-CMV-Luc2-IRES-ZsGreen-W
    suggested: RRID:Addgene_164432)
    pHDM-Hgpm2
    suggested: RRID:Addgene_164441)
    pRC-CMV-rev1b
    suggested: RRID:Addgene_164443)
    pHDM-tat1b
    suggested: RRID:Addgene_164442)
    Cell-cell fusion assay: HEK293T cells were transfected with plasmids expressing hACE2 or co-transfected with plasmids expressing SARS-COV-2 S protein (WT or mutants as mentioned) and Green Fluorescence Protein (GFP).
    hACE2
    suggested: RRID:Addgene_1786)
    Software and Algorithms
    SentencesResources
    Bioinformatics and structural analysis: Multiple sequence alignment was performed in Bioedit software using clustalW multiple alignment function.
    clustalW
    suggested: (ClustalW, RRID:SCR_017277)
    Multiple sequence alignment data was used to calculate amino acid identity matrix in Bioedit for HR1 and HR2 regions.
    Bioedit
    suggested: (BioEdit, RRID:SCR_007361)
    Identified regions are aligned and logoplots were generated using WebLogo website (
    WebLogo
    suggested: (WEBLOGO, RRID:SCR_010236)
    Structural alignment was performed in Pymol using specific PDB files of post-fusion spike protein structures of different human infecting coronaviruses (PDB id: 6LXT,1WYY,4NJL,5YL9 and 2IEQ).
    Pymol
    suggested: (PyMOL, RRID:SCR_000305)
    Molecular Dynamics Simulations: The HR1-HR2 peptide complexes harboring either WT or different mutant variants of the HR2, were subjected to molecular dynamics simulations for a period of 100 ns using Gromacs software (Abraham et al., 2015).
    Gromacs
    suggested: (GROMACS, RRID:SCR_014565)
    MMGBSA values were then plotted using Origin software.
    Origin
    suggested: (Origin, RRID:SCR_014212)
    Ligand PDB structures were built up using Chem 3D Pro or procured as SDF file data bases like PubChem, Drugbank online etc.
    PubChem
    suggested: (PubChem, RRID:SCR_004284)
    Drugbank
    suggested: (DrugBank, RRID:SCR_002700)
    Kollman charges were calculated with AutoDock Tools (ADT)
    AutoDock
    suggested: (AutoDock, RRID:SCR_012746)
    Grid potential maps were calculated using module AutoGrid 4.0.
    AutoGrid
    suggested: (Autogrid, RRID:SCR_015982)
    The 2D-interactions were interpreted from the image obtained through LigPlot v2.2.4(Wallace et al., 1995).
    LigPlot
    suggested: (LigPlot+, RRID:SCR_018249)
    36 h post-transfection both the cell lines were trysinised and co-cultured in 1:1 ratio for 12 h followed by fixing permialization and subsequent imaging using fluorescence microscope (Leica Microsystems) and subsequent analysis by ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Graphs are plotted using Microsoft Excel and Origin and represented as mean standard deviations (n = 3).
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)

    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:
    While these peptides show high efficacy in inhibiting virus replication, their widespread implementation as antiviral drugs will be challenging owing to the inherent limitations of peptide-based therapeutics (Fosgerau & Hoffmann, 2015; Otvos & Wade, 2014). On the other hand, bioavailable small molecule fusion inhibitors, which are both easier to produce and deliver than peptides, have been disappointingly scarce and only a handful have been described for IAV and HIV (Dongen et al., 2019; Zhoua et al., 2011) and more recently against CoVs (Hu et al., 2021; Park et al., 2022; Yang et al., 2020). Successful targeting of viral infection with small molecules requires identification of a suitable “molecular target” possessing the following characteristics, (i) it should represent a structural element or a sequential motif or a combination of both, which would offer a suitable interaction interface for the small molecule; (ii) the target region should be critical for the activity of the viral protein and (iii) it should be minimally tolerant towards evolutionary changes acquired through antigenic drifts or shifts. Considering these parameters, in this study, we identified a highly conserved motif, E1182-L1186-L1193 present within the HR2 helix of SARS-CoV-2 S protein, whose side chains participate in essential molecular interactions indispensable for the stability of the 6-HB (Figure 1 B, C, E and F). MD simulation analysis suggested that mutation of these residues perturb the HR1-H...

    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.

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  4. Version published to 10.1101/2022.03.16.484554v1 on bioRxiv