Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

  1. Shuai Xia
  2. Meiqin Liu
  3. Chao Wang
  4. Wei Xu
  5. Qiaoshuai Lan
  6. Siliang Feng
  7. Feifei Qi
  8. Linlin Bao
  9. Lanying Du
  10. Shuwen Liu
  11. Chuan Qin
  12. Fei Sun
  13. Zhengli Shi
  14. Yun Zhu
  15. Shibo Jiang
  16. Lu Lu

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Abstract

The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell–cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examined, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.

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  1. ScreenIT

    SciScore for 10.1101/2020.03.09.983247: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationAfter incubation, five fields were randomly selected in each well to count the number of fused and unfused cells under an inverted fluorescence microscope (Nikon Eclipse Ti-S)
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableMouse infection studies: Newborn mice were bred from pregnant mice purchased from the Animal Center of Fudan University, and all the related experiments were carried out in strict accordance with institutional regulations (approval number 20190221-070, approval date 21 February 2019).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cell Lines, viruses and Peptides: The human primary embryonic kidney cell line (293T) (CRL-3216™), Calu-3 (HTB-55™), A549 (
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    CCL-185), Vero E6 (CRL-1586™), RD (CCL-136™), and LLC-MK2 Original (CCL-7™) cells were obtained from the American Type Culture Collection (ATCC)
    Vero E6
    suggested: None
    ATCC strain of Human coronavirus 229E (HCoV-OC43, VR-740), as well as Human coronavirus OC43 (HCoV-229E, VR-1558) and HCoV-NL63 (Amsterdam strain) strains were amplified in Huh-7, HCT-8 and LLC-MK2 cells, respectively.
    HCT-8
    suggested: ICLC Cat# HTL99005, RRID:CVCL_2478)
    LLC-MK2
    suggested: BCRC Cat# 60092, RRID:CVCL_3009)
    In brief, Huh-7 cells (for testing all coronaviruses) or 293T/ACE2 cells (for testing SARS-CoV-2) were used as target cells.
    293T/ACE2
    suggested: RRID:CVCL_YZ65)
    Inhibition of pseudotyped HCoV infection: 293T cells were cotransfected with pNL4-3.luc.
    293T
    suggested: None
    To detect the inhibitory activity of a peptide on infection of coronavirus PsV, target cells (293T/ACE2 for SARS-CoV-2, SARS-CoV and SL-CoVs; RD cells for HCoV-OC43; Huh-7 for other CoVs) were plated at a density of 104 cells per well in a 96-well plate one day prior to infection 14.
    Huh-7
    suggested: None
    To test the effect of peptide on HCoV-OC43, HCoV-229E and HCoV-NL63 replication, 50 μL of 100 TCID50 virus were mixed with an equal volume of peptide and incubated at 37 ° C for 1 hour.
    HCoV-229E
    suggested: None
    Body weight and survival of the remaining six mice in each group were monitored for 14 days 35 Cytotoxicity assay: Cytotoxicity of the peptides to the cells (Vero-E6, Huh-7, LLC-MK2 and RD cells) was tested by using the Cell Counting Kit-8 (CCK-8).
    RD
    suggested: CLS Cat# 300401/p527_RD, RRID:CVCL_1649)
    Experimental Models: Organisms/Strains
    SentencesResources
    For preparing effector cells expressing S protein a coronavirus, 293T cells were transfected with one of the S protein expression vectors, including 293T/SARS-CoV-2/GFP, 293T/MERS-CoV/GFP, 293T/HCoV-229E/GFP, 293T/SARS-CoV/GFP, or 293T/SL-CoV/GFP, 293T/HCoV-OC43/GFP, 293T/HCoV-NL63/GFP or empty plasmid pAAV-IRES-EGFP.
    293T/SARS-CoV-2/GFP , 293T/MERS-CoV/GFP , 293T/HCoV-229E/GFP , 293T/SARS-CoV/GFP
    suggested: None
    Mouse infection studies: Newborn mice were bred from pregnant mice purchased from the Animal Center of Fudan University, and all the related experiments were carried out in strict accordance with institutional regulations (approval number 20190221-070, approval date 21 February 2019).
    Newborn
    suggested: None
    Software and Algorithms
    SentencesResources
    A native set of X-ray diffraction data was collected with the R-AXIS IV ++ detector (Rigaku, Japan) with an exposure time of 3 min per image and was indexed and processed using iMosflm 36.
    iMosflm
    suggested: (iMosflm , RRID:SCR_014217)
    The final model was manually adjusted in COOT and refined with Refmac 38.
    COOT
    suggested: (Coot, RRID:SCR_014222)
    The interaction model of EK1C4 peptide and HR1 domains of SARS-nCoV-2 was predicted by SWISS-MODEL sever 39 using 6XLT as reference for EK1 moiety, and by Autodock 4 software 40 for cholesterol moiety (Fig. S8).
    Autodock
    suggested: (AutoDock, RRID:SCR_012746)
    Statistical analysis: The survival rates of mice were analyzed by GraphPad Prism 5.0 software.
    GraphPad Prism
    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 33 and 35. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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  2. Version published to 10.1038/s41422-020-0305-x
  3. Version published to 10.1101/2020.03.09.983247v1 on bioRxiv