Niclosamide inhibits SARS-CoV2 entry by blocking internalization through pH-dependent CLIC/GEEC endocytic pathway

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

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Many viruses utilize the host endo-lysosomal network to infect cells. Tracing the endocytic itinerary of SARS-CoV2 can provide insights into viral trafficking and aid in designing new therapeutic targets. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV2 is internalized via the clathrin and dynamin-independent, pH-dependent CLIC/GEEC (CG) endocytic pathway. Endosomal acidification inhibitors like BafilomycinA1 and NH 4 Cl, which inhibit the CG pathway, strongly block the uptake of RBD. Using transduction assays with SARS-CoV2 Spike-pseudovirus, we confirmed that these acidification inhibitors also impede viral infection. By contrast, Chloroquine neither affects RBD uptake nor extensively alters the endosomal pH, yet attenuates Spike-pseudovirus entry, indicating a pH-independent mechanism of intervention. We screened a subset of FDA-approved acidification inhibitors and found Niclosamide to be a potential SARS-CoV2 entry inhibitor. Niclosamide, thus, could provide broader applicability in subverting infection of similar category viruses entering host cells via this pH-dependent endocytic pathway.

Article activity feed

  1. 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.

  2. Version published to 10.1101/2020.12.16.422529 on bioRxiv