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. 2020 Dec;9(1):2245-2255.
doi: 10.1080/22221751.2020.1829082.

Targeting the endolysosomal host-SARS-CoV-2 interface by clinically licensed functional inhibitors of acid sphingomyelinase (FIASMA) including the antidepressant fluoxetine

Sebastian Schloer  1   2 , Linda Brunotte  3   2 , Jonas Goretzko  1   2 , Angeles Mecate-Zambrano  3   2 , Nadia Korthals  1 , Volker Gerke  1   2 , Stephan Ludwig  3   2 , Ursula Rescher  1   2
Affiliations

Targeting the endolysosomal host-SARS-CoV-2 interface by clinically licensed functional inhibitors of acid sphingomyelinase (FIASMA) including the antidepressant fluoxetine

Sebastian Schloer et al. Emerg Microbes Infect. 2020 Dec.

Abstract

The Coronavirus 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 severity. 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.

Keywords: FIASMA; IAV; SARS-CoV-2; endolysosomal interference; fluoxetine; viral entry.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Antiviral potential of fluoxetine treatment against IAV subtypes pdm09 and Panama in Calu-3 cells. (A) Virus titres determined in Calu-3 cells infected with the respective IAV subtype at 0.01 MOI for 24 h. Cells were pretreated with solvent or fluoxetine for 16 h. Data points present mean virus titres ± SEM of three independent experiments. (B) Released viral titres normalized to the control condition and log-transformed fluoxetine concentrations were used to generate the dose-response curves. EC50 and EC90 values were determined using the 4PL nonlinear regression model.
Figure 2.
Figure 2.
Analysis of anti-SARS-CoV-2 activities of fluoxetine and U18666A treatment in Vero E6 cells and Calu-3 cells. (A) Virus titres determined in Calu-3 and Vero E6 cells infected with SARS-CoV-2 at 0.1 MOI for 48 h. Treatment of infected cells with solvent or fluoxetine was started 1 h p.i. Data points present mean virus titres ± SEM of three independent experiments. (B) To generate the dose-response curves, virus release was normalized to the control condition, fluoxetine concentrations were log-transformed, and nonlinear regression and a 4PL model was used to fit the curves and to determine the EC50 and EC90 values. (C) Polarized Calu-3 cells grown on semipermeable supports were infected with SARS-CoV-2 isolate at 0.1 MOI for 48 h. Cells were treated 1 h p.i. with 20 µM fluoxetine, and 2 or 10 µg/mL U18666A. Bar graphs represent the mean viral titres ± SEM of three independent experiments. One-way ANOVA followed by by Dunnett’s multiple comparison test. **p ≤ .01, ***p ≤ .001.
Figure 3.
Figure 3.
Amiodarone and imipramine as two classic representative of the FIASMA group reduced SARS-CoV-2 and IAV Panama titre. Virus titres determined in Calu-3 cells infected with (A) SARS-CoV-2 at 0.1 MOI for 48 h or (B) with the IAV strain Panama at 0.01 MOI for 24 h. Treatment of infected cells with solvent or amiodarone (5 µM) or imipramine (50 µM) was started 1 h p.i. Data points present mean virus titres ± SEM of three independent experiments. (C) Analysis of cell viability. MTT assay of Calu-3 cells treated with the solvent DMSO (C), amiodarone (5 µM) or imipramine (50 µM) for 48 h. The protein kinase inhibitor staurosporine (ST), a strong inducer of cytotoxicity, served as a positive control. Bar graphs represent the mean viral titres ± SEM of three independent experiments. One-way ANOVA followed by Dunnett’s multiple comparison test; **p ≤ .01, ****p ≤ .0001.
Figure 4.
Figure 4.
Increased endolysosomal cholesterol storage and dysregulated acidification upon fluoxetine treatment. Vero E6 cells were treated for 16 h with either the solvent DMSO, fluoxetine, or U18666A at the indicated concentrations. (A) Representative 2D maximum intensity projections of entire z-stacks obtained by confocal imaging. LELs were identified via immunodetection of the LEL marker protein CD63 and unesterified cholesterol was visualized using filipin. Representative pseudocoloured digital images are shown. To generate the heat maps, filipin-positive pixels were colour-encoded according to their intensity values. Scale bar, 20 µm. (B) For each cell, the colocalization of filipin with CD63 quantitated across the entire z-stack was calculated as Manders’ coefficient. Bar graphs represent means ± SEM of 3 individual cells per condition from three independent experiments. (C) Endolysosomal pH values in Calu-3 cells were measured by ratio imaging. Bar graphs present mean pH values ± SEM of 56 cells for each condition from three independent experiments. One-way ANOVA followed by Dunnett’s multiple comparison test; ns, not significant, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001.
Figure 5.
Figure 5.
Impact of fluoxetine and U18666A on SARS-CoV-2 infection success within the first cycle of replication. (A) Vero E6 and (B) Calu-3 cells preatreated with the drugs at the indicated concentrations were infected with SARS-CoV-2 at 1 MOI for 1 h. Nuclei were visualized with DAPI. To determine infection rates, NP-positive cells were detected by immunofluorescence imaging. Mean percentages ± SEM of NP-positive cells were calculated from three independent experiments. One-way ANOVA followed by Dunnett’s multiple comparison test. ****p ≤ .0001.
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Grants and funding

This research was funded by grants from German Research Foundation (DFG), CRC1009 “Breaking Barriers,” Project A06 (to U.R.) and B02 (to S.L.), CRC1348 “Dynamic Cellular Interfaces,” Project A11 (to U.R.), KFO342 TP6, Br5189/3-1 (to L.B.), Lu477/30-1 (to S.L.), Interdisciplinary Center for Clinical Research (IZKF) of the Münster Medical School [grant number Re2/022/20] and from the Innovative Medizinische Forschung (IMF) of the Münster Medical School [grant number SC121912] (to S.S.).