Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors

  1. Chaitra Prabhakara
  2. Rashmi Godbole
  3. Parijat Sil
  4. Sowmya Jahnavi
  5. Shah-e-Jahan Gulzar
  6. Thomas S. van Zanten
  7. Dhruv Sheth
  8. Neeraja Subhash
  9. Anchal Chandra
  10. Akshatha Shivaraj
  11. Patricia Panikulam
  12. Ibrahim U
  13. Vijay Kumar Nuthakki
  14. Theja Parassini Puthiyapurayil
  15. Riyaz Ahmed
  16. Ashaq Hussain Najar
  17. Sai Manoz Lingamallu
  18. Snigdhadev Das
  19. Bhagyashri Mahajan
  20. Praveen Vemula
  21. Sandip B. Bharate
  22. Parvinder Pal Singh
  23. Ram Vishwakarma
  24. Arjun Guha
  25. Varadharajan Sundaramurthy
  26. Satyajit Mayor

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH 4 Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.

Article activity feed

  1. Version published to 10.1371/journal.ppat.1009706
  2. ScreenIT

    SciScore for 10.1101/2020.12.16.422529: (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
    To label surface ACE2, fixed cells were blocked with 10mg/ml bovine serum albumin (30 minutes) followed by incubation with anti-myc primary antibody (1 hour) and secondary antibody (45 minutes) in blocking buffer at RT.
    ACE2
    suggested: None
    anti-myc
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Spike-pseudovirus transduction assays: AGS/HEK-293T cells were plated in optical bottom 96-well plates.
    AGS/HEK-293T
    suggested: None
    In the case of HEK-293T cells (Figure S10G), MTT cell viability assay was performed to check toxicity (assay described in Supplementary Methods).
    HEK-293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Imaging and Analysis: Statistical methods and hypothesis testing: All statistical tests between control and treatment were performed in MATLAB using Wilcoxon rank-sum test and the p-value of the hypothesis testing and the number of repeats is indicated in figure legends.
    MATLAB
    suggested: (MATLAB, RRID:SCR_001622)

    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.

    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.

  3. Version published to 10.1101/2020.12.16.422529v1 on bioRxiv