The SARS-CoV-2 Cytopathic Effect Is Blocked by Lysosome Alkalizing Small Molecules

  1. Kirill Gorshkov
  2. Catherine Z. Chen
  3. Robert Bostwick
  4. Lynn Rasmussen
  5. Bruce Nguyen Tran
  6. Yu-Shan Cheng
  7. Miao Xu
  8. Manisha Pradhan
  9. Mark Henderson
  10. Wei Zhu
  11. Eunkeu Oh
  12. Kimihiro Susumu
  13. Mason Wolak
  14. Khalida Shamim
  15. Wenwei Huang
  16. Xin Hu
  17. Min Shen
  18. Carleen Klumpp-Thomas
  19. Zina Itkin
  20. Paul Shinn
  21. Juan Carlos de la Torre
  22. Anton Simeonov
  23. Sam G. Michael
  24. Matthew D. Hall
  25. Donald C. Lo
  26. Wei Zheng

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Abstract

Understanding the SARS-CoV-2 virus’ pathways of infection, virus–host–protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. Therefore, we evaluated additional lysosomotropic compounds to identify an alternative lysosome-based drug repurposing opportunity. We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 μM and selectivity indices (SIs; SI = CC50/EC50) ranging from 1.5- to >10-fold. The compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) reduced (ROC-325) viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings suggest the lysosome as a potential host cell target to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.

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  1. Version published to 10.1021/acsinfecdis.0c00349
  2. ScreenIT

    SciScore for 10.1101/2020.05.16.091520: (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
    LysoTracker Deep Red (L12492), goat-anti-mouse AlexaFluor-647 (A-21242, RRID:AB_2535811), HCS Cell Mask Green (H32714)
    goat-anti-mouse AlexaFluor-647
    detected: (Thermo Fisher Scientific Cat# A-21242, RRID:AB_2535811)
    LC3B primary rabbit antibody (3868S, RRID:AB_2137707) was purchased from Cell Signaling Technologies. Cell Staining Buffer (420201) was purchased from BioLegend.
    LC3B
    detected: (Cell Signaling Technology Cat# 3868, RRID:AB_2137707)
    Plates were then incubated with rabbit-anti-LC3B (Cell Signaling Technologies, Danvers, MA) antibodies in Cell Staining Buffer for 2 h at room temperature.
    rabbit-anti-LC3B ( Cell Signaling Technologies , Danvers , MA )
    suggested: None
    Plates were washed three times with PBS and secondary antibody goat-anti-mouse AlexaFluor-647 (Invitrogen) were added in Cell Staining Buffer for 1 h.
    AlexaFluor-647 (Invitrogen)
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The following items were purchased from ATCC: EMEM (30-2003), Vero-E6 (CRL-1586, RRID:CVCL_0574), HeLa (CCL-2, RRID:CVCL_0030)
    ATCC: EMEM ( 30-2003 ) , Vero-E6 ( CRL-1586
    detected: (IZSLER Cat# BS CL 87, RRID:CVCL_0574)
    HeLa
    detected: (BCRC Cat# 60005, RRID:CVCL_0030)
    , HEK293T (CRL-3216, RRID:CVCL_0063).
    HEK293T
    detected: (CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Cell Culture: Vero-E6 cells previously selected for high ACE2 expression (82) were cultured in MEM/10% HI FBS supplemented with 0.5 μg/mL amphotericin B and passaged twice per week at 1:5 dilutions using trypsin.
    Vero-E6
    suggested: None
    , HeLa CCL-2, HEK293T and Huh-7.5 (grown in DMEM, 10% FBS, and 1% Penicillin/Streptomycin) were cultured in T175 flasks and passaged at 95% confluency.
    HeLa
    suggested: None
    HEK293T
    suggested: None
    Huh-7.5
    suggested: RRID:CVCL_7927)
    The plates were transported into the BSL-3 facility were a 25 μL aliquot of virus inoculated cells (4000 Vero E6 cells/well) was added to each well in columns 3-24.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    TrypLE (12604013), PBS -/- (w/o Ca2+ or Mg2+) (
    PBS -/-
    suggested: None
    Vero-E6 (grown in EMEM, 10% FBS, and 1% Penicillin/Streptomycin)
    Vero-E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Image montages were prepared using Fiji (ImageJ, NIH).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    High-content image analysis data was downloaded as a Microsoft Excel spreadsheet.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    EC50 values were obtained using non-linear regression in Graphpad Prism 7.04.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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, there are limitations to the CPE assay including its dependence on the host response and the fact that it is an indirect measurement of SARS-CoV-2 infection and replication. The phenotypic outcome can also vary depending on culture conditions and viral multiplicity of infection (MOI), number of virions that are added per cell during infection (58). The potencies of drug protection against virally-induced cell death can be lower than in other assays that directly measure viral load. Nevertheless, this study confirms that SARS-CoV-2 infection in Vero-E6 cells results in cell death similar to other reports, and that CPE can be suppressed by blocking autophagy with small molecule inhibitors to the same extent as positive control remdesivir (59, 60). Recently, a drug-repurposing screen of FDA-approved compounds, using a similar CPE assay with SARS-CoV-2 in Vero-E6 cells, found clomipramine (IC50 5.93 μM; CC50 >30 μM) and mefloquine (IC50 7.11 μM; CC50 >18.5 μM) to be active with low toxicity (61). The same study found HCQ to be more active than CQ with an IC50 of 9.21 μM and 42.03 μM, respectively. Mefloquine was also found to be active in another SARS-CoV-2 CPE screen using Caco-2 cells with an IC50 of 14.1 μM (62). In our study, the SI was calculated using the ratio of the EC50, the half-maximal effective concentration, and the CC50, the half-maximal cytotoxic concentration. Between the CPE and the autophagy assays there was good correspondence in the cytotoxicity measu...

    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 10.1101/2020.05.16.091520 on bioRxiv