Prunella vulgaris extract and suramin block SARS-coronavirus 2 virus Spike protein D614 and G614 variants mediated receptor association and virus entry in cell culture system

  1. Zhujun Ao
  2. Mable Chan
  3. Maggie Jing Ouyang
  4. Olukitibi Titus Abiola
  5. Mona Mahmoudi
  6. Darwyn Kobasa
  7. Xiaojian Yao

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Abstract

Until now, no approved effective vaccine and antiviral therapeutic are available for treatment or prevention of SARS-coronavirus 2 (SCoV-2) virus infection. In this study, we established a SCoV-2 Spike glycoprotein (SP), including a SP mutant D614G, pseudotyped HIV-1-based vector system and tested their ability to infect ACE2-expressing cells. This study revealed that a C-terminal 17 amino acid deletion in SCoV-2 SP significantly increases the incorporation of SP into the pseudotyped viruses and enhanced its infectivity, which may be helpful in the design of SCoV2-SP-based vaccine strategies. Moreover, based on this system, we have demonstrated that an aqueous extract from the Chinese herb Prunella vulgaris (CHPV) and a compound, suramin, displayed potent inhibitory effects on both wild type and mutant (G614) SCoV-2 SP pseudotyped virus (SCoV-2-SP-PVs)-mediated infection. The 50% inhibitory concentration (IC50) for CHPV and suramin on SCoV-2-SP-PVs are 30, and 40 μg/ml, respectively. To define the mechanisms of their actions, we demonstrated that both CHPV and suramin are able to directly interrupt SCoV-2–SP binding to its receptor ACE2 and block the viral entry step. Importantly, our results also showed that CHPV or suramin can efficiently reduce levels of cytopathic effect caused by SARS-CoV-2 virus (hCoV-19/Canada/ON-VIDO-01/2020) infection in Vero cells. Furthermore, our results demonstrated that the combination of CHPV/suramin with an anti-SARS-CoV-2 neutralizing antibody mediated more potent blocking effect against SCoV2-SP-PVs. Overall, this study provides evidence that CHPV and suramin has anti-SARS-CoV-2 activity and may be developed as a novel antiviral approach against SARS-CoV-2 infection.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.28.270306: (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
    The rabbit polyclonal antibody against SARS-CoV-2 SP (Cat# 40150-R007) and ACE2 protein (Cat# 40592-T62) were obtained from Sino Biological and anti-HIVp24 monoclonal antibody was described previously [35,36].
    SARS-CoV-2
    suggested: (Sino Biological Cat# 40150-R007, RRID:AB_2827979)
    SP
    suggested: None
    anti-HIVp24
    suggested: None
    neutralizing Antibody (nAb) Human IgG1(SAD-535) was purchased from ACRO Biosystems..
    neutralizing Antibody ( nAb ) Human IgG1 ( SAD-535 )
    suggested: None
    Human IgG1
    suggested: None
    After incubation for 3 hours, wells were washed three times and a goat anti-ACE2 antibody that binds to the Spike-ACE2 complex was added followed by applying the HRP-conjugated anti-goat IgG and 3,3’,5,5’-tetramethylbenzidine (TMB) substrate.
    anti-ACE2
    suggested: None
    anti-goat IgG
    suggested: None
    Western blot (WB) analyses: To detect cellular protein ACE2, SARS-CoV-2-SP, or SPΔC in transfected cells or SCoV-2-SP-VPs, transfected 293TACE2 cells or VPs were lysed in RIPA buffer, and directly loaded into the 10 % SDS–PAGE gel and the presence of each protein was detected by WB with various corresponding antibodies.
    SARS-CoV-2-SP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture, antibodies and chemicals: The human embryonic kidney cells (HEK293T) and kidney epithelial cells (VeroE6 and Vero cells (ATCC, CCL-81)) from African green monkey were cultured in Dulbecco’s modified Eagle’s medium (HEK293T, VeroE6) or Minimum Essential Medium (MEM; Vero)
    Vero
    suggested: None
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Western blot (WB) analyses: To detect cellular protein ACE2, SARS-CoV-2-SP, or SPΔC in transfected cells or SCoV-2-SP-VPs, transfected 293TACE2 cells or VPs were lysed in RIPA buffer, and directly loaded into the 10 % SDS–PAGE gel and the presence of each protein was detected by WB with various corresponding antibodies.
    293TACE2
    suggested: RRID:CVCL_YZ65)

    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:
    However, one disadvantage of using nAb as an anti-SARS-COV-2 agent is its source limitation. Therefore, the finding of the synergistic effect of a combination of nAb with other agents, such as CHPV or Suramin is beneficial for (i) similar efficiencies would be achieved by using reduced amounts of antibody and CHPV or Suramin, (ii) the combination of nAb and CHPV/suramin will reduce the likelihood of viral resistance. Whether these enhanced effects might be due to a combined effect through their different binding mechanisms still needs to be investigated. The effectiveness of CHPV and/or suramin against SARS-COV-2 infection in vivo remains to be investigated. Our findings could be further validated in an appropriate animal model and clinical trials for prevention of COVID-19. Since SARS-COV-2 infection initiates in the respiratory tract [43], the use of CHPV and/or Suramin as nasopharynx agents (Nasal spray) to prevent initial SARS-COV-2 infection and transmission in the respiratory tract will be a particularly attractive strategy, and will require further efficacy studies. Overall, we demonstrated that CHPV and suramin possess an anti-SARS-COV-2 entry inhibitor activity and functions at least partially by interrupting SARS-COV-2 SP binding to its receptor. Additional in vivo safety and protection studies will facilitate its application as an option to help control the ongoing SARS-CoV-2 pandemic.

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.08.28.270306v1 on bioRxiv