Targeting the endolysosomal host-SARS-CoV-2 interface by clinically licensed functional inhibitors of acid sphingomyelinase (FIASMA) including the antidepressant fluoxetine
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Abstract
The Corona Virus Disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Related Coronavirus 2 (SARS-CoV-2) is a global health emergency. As only very limited therapeutic options are clinically available, there is an urgent need for the rapid development of safe, effective, and globally available pharmaceuticals that inhibit SARS-CoV-2 entry and ameliorate COVID-19. In this study, we explored the use of small compounds acting on the homeostasis of the endolysosomal host-pathogen interface, to fight SARS-CoV-2 infection. We find that fluoxetine, a widely used antidepressant and a functional inhibitor of acid sphingomyelinase (FIASMA), efficiently inhibited the entry and propagation of SARS-CoV-2 in the cell culture model without cytotoxic effects and also exerted potent antiviral activity against two currently circulating influenza A virus subtypes, an effect which was also observed upon treatment with the FIASMAs amiodarone and imipramine. Mechanistically, fluoxetine induced both impaired endolysosomal acidification and the accumulation of cholesterol within the endosomes. As the FIASMA group consists of a large number of small compounds that are well-tolerated and widely used for a broad range of clinical applications, exploring these licensed pharmaceuticals may offer a variety of promising antivirals for host-directed therapy to counteract enveloped viruses, including SARS-CoV-2 and COVID 19.
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SciScore for 10.1101/2020.07.27.222836: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis Data analysis: A priori power analysis (G*Power 3.1 37) was used to estimate the required sample sizes. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Late endosomal/lysosomal compartments were identified by staining the endolysosomal marker protein CD63 with the mouse monoclonal anti-CD63 antibody H5C6 (1:300 in 2% BSA in PBS) for 90 min. anti-CD63suggested: NoneThe secondary AlexaFluor594-coupled anti-mouse antibody was from Thermo Fisher Limited. anti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Reso… SciScore for 10.1101/2020.07.27.222836: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis Data analysis: A priori power analysis (G*Power 3.1 37) was used to estimate the required sample sizes. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Late endosomal/lysosomal compartments were identified by staining the endolysosomal marker protein CD63 with the mouse monoclonal anti-CD63 antibody H5C6 (1:300 in 2% BSA in PBS) for 90 min. anti-CD63suggested: NoneThe secondary AlexaFluor594-coupled anti-mouse antibody was from Thermo Fisher Limited. anti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources Cells and treatment: The human bronchioepithelial cell line Calu-3, the Madin-Darby canine kidney (MDCK) II cells, and the Vero E6 cell line were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% standardized fetal bovine serum (FBS Advance; Capricorne), 2 mM L-glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. MDCKsuggested: NoneCytotoxicity assay: Calu-3 and Vero cells were treated at the indicated concentrations with the solvent DMSO, U18666A, fluoxetine, amiodarone and imipramine. Verosuggested: NoneIn brief, MDCK cells (for IAV infection) or Vero E6 cells (for SARS-CoV-2 infection) grown to a monolayer in six-well dishes were washed with PBS and infected with serial dilutions of the respective supernatants in infection-PBS for 1 hour at 37°C. Vero E6suggested: NoneSingle-cycle infection assay: Calu-3 cells were infected with SARS-CoV2 and fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature at the indicated times p.i. Calu-3suggested: NoneSoftware and Algorithms Sentences Resources The secondary AlexaFluor594-coupled anti-mouse antibody was from Thermo Fisher Limited. Thermo Fisher Limitedsuggested: NoneThe colocalization of filipin and CD63 signals was analyzed using the JACoP plugin 35 for Fiji 36. Fijisuggested: (Fiji, RRID:SCR_002285)Data analysis: A priori power analysis (G*Power 3.1 37) was used to estimate the required sample sizes. G*Powersuggested: (G*Power, RRID:SCR_013726)Data were analyzed with Prism 8.00 (Graph-Pad). Prismsuggested: (PRISM, RRID:SCR_005375)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 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.
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SciScore for 10.1101/2020.07.27.222836: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis A priori power analysis (G*Power 3.1(34)) was used to estimate the required sample sizes. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Late endosomal/lysosomal compartments were identified by staining the endolysosomal marker protein CD63 with the mouse monoclonal anti-CD63 antibody H5C6 (1:300 in 2% BSA in PBS) for 90 min. anti-CD63suggested: NoneThe secondary AlexaFluor594-coupled anti-mouse … SciScore for 10.1101/2020.07.27.222836: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis A priori power analysis (G*Power 3.1(34)) was used to estimate the required sample sizes. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Late endosomal/lysosomal compartments were identified by staining the endolysosomal marker protein CD63 with the mouse monoclonal anti-CD63 antibody H5C6 (1:300 in 2% BSA in PBS) for 90 min. anti-CD63suggested: NoneThe secondary AlexaFluor594-coupled anti-mouse antibody was from Thermo Fisher Limited. anti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources Calu-3 and Vero cells were treated at the indicated concentrations with the solvent DMSO, U18666A, or fluoxetine. Calu-3suggested: BCRJ Cat# 0264, RRID:CVCL_0609Verosuggested: NoneIn brief, Vero E6 cells grown to a monolayer in six-well dishes were washed with PBS and infected with serial dilutions of the respective supernatants in infection-PBS for 1 hour at 37°C. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources The secondary AlexaFluor594-coupled anti-mouse antibody was from Thermo Fisher Limited. Thermo Fisher Limitedsuggested: (DataAssist, RRID:SCR_014969)A priori power analysis (G*Power 3.1(34)) was used to estimate the required sample sizes. G*Powersuggested: (G*Power, RRID:SCR_013726)Data were analyzed with Prism 8.00 (Graph-Pad). Prismsuggested: (PRISM, RRID:SCR_005375)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 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.
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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.
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SciScore for 10.1101/2020.07.27.222836: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis A priori power analysis (G*Power 3.1(34)) was used to estimate the required sample sizes. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Late endosomal/lysosomal compartments were identified by staining the endolysosomal marker protein CD63 with the mouse monoclonal anti-CD63 antibody H5C6 (1:300 in 2% BSA in PBS) for 90 min. anti-CD63suggested: NoneThe secondary AlexaFluor594-coupled anti-mouse … SciScore for 10.1101/2020.07.27.222836: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis A priori power analysis (G*Power 3.1(34)) was used to estimate the required sample sizes. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Late endosomal/lysosomal compartments were identified by staining the endolysosomal marker protein CD63 with the mouse monoclonal anti-CD63 antibody H5C6 (1:300 in 2% BSA in PBS) for 90 min. anti-CD63suggested: NoneThe secondary AlexaFluor594-coupled anti-mouse antibody was from Thermo Fisher Limited. anti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources Calu-3 and Vero cells were treated at the indicated concentrations with the solvent DMSO, U18666A, or fluoxetine. Calu-3suggested: BCRJ Cat# 0264, RRID:CVCL_0609Verosuggested: NoneCalu-3 cells and Vero E6 cells were infected with the SARS-CoV-2 isolate hCoV-19/Germany/FI1103201/2020 (EPI-ISL_463008, mutation D614G in spike protein) in infection-PBS (containing 0.2% BSA, 1% CaCl2, 1% MgCl2, 100 U/mL penicillin and 0.1 mg/mL streptomycin) at MOI 0.1 for 1 hour. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources The secondary AlexaFluor594-coupled anti-mouse antibody was from Thermo Fisher Limited. Thermo Fisher Limitedsuggested: (DataAssist, RRID:SCR_014969)A priori power analysis (G*Power 3.1(34)) was used to estimate the required sample sizes. G*Powersuggested: (G*Power, RRID:SCR_013726)Data were analyzed with Prism 8.00 (Graph-Pad). Prismsuggested: (PRISM, RRID:SCR_005375)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 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.
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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.
-
SciScore for 10.1101/2020.07.27.222836: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis A priori power analysis (G*Power 3.1(34)) was used to estimate the required sample sizes. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Late endosomal/lysosomal compartments were identified by staining the endolysosomal marker protein CD63 with the mouse monoclonal anti-CD63 antibody H5C6 (1:300 in 2% BSA in PBS) for 90 min. anti-CD63suggested: NoneThe secondary AlexaFluor594-coupled anti-mouse antibody was from Thermo Fisher Limited. … SciScore for 10.1101/2020.07.27.222836: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis A priori power analysis (G*Power 3.1(34)) was used to estimate the required sample sizes. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Late endosomal/lysosomal compartments were identified by staining the endolysosomal marker protein CD63 with the mouse monoclonal anti-CD63 antibody H5C6 (1:300 in 2% BSA in PBS) for 90 min. anti-CD63suggested: NoneThe secondary AlexaFluor594-coupled anti-mouse antibody was from Thermo Fisher Limited. anti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources In this study, we explored the impact of disturbed ASMase activity and endolysosomal cholesterol accumulation on SARS-CoV-2 infection in Vero E6 cells commonly used to investigate SARS-CoV-2 infection, and in polarized bronchial Calu-3 cell monolayers, a lung cell model for productive SARS-CoV-2 infection. Vero E6suggested: CVCL_XD71Results Fluoxetine treatment impairs SARS-CoV-2 infection in Calu-3 and Vero cells To assess the antiviral potential of fluoxetine, we treated SARS-CoV-2 infected Vero and Calu3 cells, two cell lines that are known to produce infectious virus progeny upon SARS-CoV-2 exposure, 1 h p.i. with a range of fluoxetine concentrations and measured the resulting virus titers 48 h p. Verosuggested: NoneFluoxetine treatment is associated with Increased endolysosomal cholesterol storage and dysregulated acidification Because the pronounced decrease in SARS-CoV-2 titers observed upon treatment with U18666A hinted at an antiviral capacity of dysregulated endolysosomal cholesterol contents in SARS-CoV-2 infection, we next determined whether fluoxetine treatment of Vero and Calu3 cells was associated with increased endolysosomal cholesterol pools. Calu3suggested: BCRJ Cat# 0264, CVCL_0609The human bronchioepithelial cell line Calu-3 and the Vero E6 cell line were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% standardized fetal bovine serum (FBS Advance; Capricorne), 2 mM L-glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Calu-3suggested: BCRJ Cat# 0264, CVCL_0609Software and Algorithms Sentences Resources Our results show that fluoxetine treatment inhibited SARS-CoV-2 infection in a dose-dependent manner up to 99% and thus support the hypothesis that the endolysosomal lipid balance is a promising druggable target at the hostvirus interface for a wide variety of enveloped viruses, including SARS-CoV-2. SARS-CoV-2suggested: (Active Motif Cat# 91345, AB_2847847)Tapping this vast pool of potential antivirals for drug repurposing might be a fast and powerful approach to fight a broad range of pathogens with functionally similar modes of action, including SARSCoV-2. SARSCoV-2suggested: NoneThe secondary AlexaFluor594-coupled anti-mouse antibody was from Thermo Fisher Limited. Thermo Fisher Limitedsuggested: (DataAssist, SCR_014969)A priori power analysis (G*Power 3.1(34)) was used to estimate the required sample sizes. G*Powersuggested: (G*Power, SCR_013726)Data were analyzed with Prism 8.00 (Graph-Pad). Prismsuggested: (PRISM, SCR_005375)Data from additional tools added to each annotation on a weekly basis.
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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.
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