only very limited therapeutic options are clinically available, there is an urgent need for the 23 rapid development of safe, effective, and globally available pharmaceuticals that inhibit SARS-24
CoV-2 entry and ameliorate COVID-19. In this study, we explored the use of small compounds 25 acting on the homeostasis of the endolysosomal host-pathogen interface, to fight SARS-CoV-26 2 infection. We find that fluoxetine, a widely used antidepressant and a functional inhibitor of 27 acid sphingomyelinase (FIASMA), efficiently inhibited the entry and propagation of SARS-CoV-28 2 in the cell culture model without cytotoxic effects and also exerted potent antiviral activity 29 against two currently circulating influenza A virus subtypes, an effect which was also observed 30 upon treatment with the FIASMAs amiodarone and imipramine. Mechanistically, fluoxetine 31 induced both impaired endolysosomal acidification and the accumulation of cholesterol within 32 the endosomes. As the FIASMA group consists of a large number of small compounds that 33 are well-tolerated and widely used for a broad range of clinical applications, exploring these 34 licensed pharmaceuticals may offer a variety of promising antivirals for host-directed therapy 35
The current outbreak of the Severe Acute Respiratory Syndrome Related Coronavirus 2 46 (SARS-CoV-2) and the resulting Corona Virus Disease 2019 (COVID-19) pandemic clearly 47 6 added to the cells for 4 h. The supernatant was aspirated, DMSO was added to the cells for 5 126 min, and subsequently, the OD562 was measured 34 . 127
Filipin staining and colocalization analysis. Cells The secondary AlexaFluor594-coupled anti-mouse antibody was from Thermo Fisher Limited. 135
Confocal microscopy was performed with an LSM 780 microscope (CarlZeiss, Jena, Germany) 136 equipped with a Plan-Apochro-mat 63x/1.4 oil immersion objective. The colocalization of filipin 137 and CD63 signals was analyzed using the JACoP plugin 35 for Fiji 36 . 138
Endosomal pH measurement. Ratiometric fluorescence microscopy and calculation of 139 endosomal pH were done as described 12 . In brief, cells were exposed to Oregon green 488 140 (OG488)-labeled and tetramethylrhodamine (TMR)-labeled dextran (Invitrogen) (10 kDa) for 141 60 min, followed by a 60 min chase. Cells were then washed and kept in HEPES-buffered 142
Hanks' balanced salt solution (HBSS) at 37°C during image acquisition. The individual 143 epifluorescence signals of each dye were acquired at intervals of 60 s, and the endosomal pH 144 values of the cells were calculated according to the calibration curve generated by applying 145 standard solutions ranging from pH 4.5 to 6.0 to cells. were evaluated using one-way ANOVA followed by Dunnett's multiple comparison test. 176 **p<0.01, ***p< 0.001, ****p ≤ 0.0001. 177
Results 178
Building on our previous work on the LEL cholesterol balance as a promising therapeutic target CoV-2 titers up to 99% in both cell lines (Fig. 2B) . reproduced in the polarized cell model. Of note, U18666A treatment was also antiviral, with a 207 lower concentration of 2 µg/mL reducing the viral titers to 75%, and the higher 10 µg/mL dose 208 displaying a more pronounced antiviral effect of a 99% reduction. Because unwanted cytotoxic 209 effects are one of the main causes for the withdrawal of approved drugs, we measured the 210 impact of U18666A and fluoxetine treatments on Calu-3 and Vero cell viability. As shown in 211
Suppl. Fig. 1 , both drug treatments were well tolerated, and cell viability was unaffected over A key feature of the late endosomal compartment is the acidic pH. We, therefore, assessed 243 whether fluoxetine treatment impacted endolysosomal pH values in Calu-3 cells, utilizing a 244 quantitative ratiometric fluorescence microscopy assay 12,13 . In control cells, the measured pH 245 was at a value of 4.7, whereas cells exposed to 10 µg/mL U18666A displayed a significantly 246 reduced acidification, well in agreement with our previously reported data of the impact of 247 higher U18666A concentrations on pH values in LELs 19 . Of note, the endolysosomal 248 acidification was also affected upon fluoxetine treatment, and this was already observed in 249 cells treated with the low 5 µM concentration ( Figure 4C) . Of note, even a low 5 µM fluoxetine treatment could reduce the number of visibly infected Vero 257 cells to 5%, and the higher dose of 20 µM reduced the levels of infected cells to 2%, indicating 258 a 90% inhibition of cells with detectable SARS-CoV-2 infection. In contrast to the dose-259 response assays, this assay does not discriminate between different signal strengths, and EC50 260 values are, therefore, not directly comparable. 261
In this study, we explored the use of small compounds that target the endolysosomal host- approved, generally well-tolerated, and widely used in human medicine for the treatment of a 300 broad spectrum of pathological conditions 16 . The FIASMA fluoxetine, trade-named Prozac, is 301 a selective serotonin reuptake inhibitor that boomed in the 1980s and 1990s in the US and is 302 commonly used to treat major depression and related disorders. Our results show that 303 fluoxetine treatment was capable of inhibiting SARS-CoV-2 infection in a dose-dependent 304 manner, with an EC50 value below 1 µM, and that the application of 10 µM fluoxetine severely 305 reduced viral titers up to 99%. Fluoxetine-mediated pH neutralization was already seen at a 306 low dose, whereas enhanced endolysosomal cholesterol pools were only visible when a higher 307 dose was used. 308
Our results support the hypothesis that although there is quite some variation in the actual 309 escape mechanisms 11 , targeting the viral entry might serve as a target for antiviral therapy 33 . 310
The intricate regulatory circuits that underly endolysosomal lipid balance and functionality are 311 key elements functioning at the endolysosomal host-virus interface and are promising 312 druggable targets for a wide variety of viruses and might be a fast and versatile approach to 313 fight a broad range of pathogens with functionally similar modes of action. Because of the 314 essential need for the host cell components, the infection cycle would have to be drastically 315 altered to circumvent such host-directed therapeutics, and this approach is therefore 316 considered much less likely to cause the development of resistance. The large variety of 317 FIASMA pharmaceuticals offer a toolbox of potential antivirals for host-directed therapy, and 318 exploring their use including their combination with drugs that directly target viral enzymes, 319 might constitute a promising approach to repurpose these drugs as antivirals to counteract 320 SARS-CoV-2 and COVID 19. Calu-3 cells grown on semipermeable supports were infected with SARS-CoV-2 isolate at 0.1 449 MOI for 48 h. Cells were treated 1 h p.i. with 20 µM fluoxetine, and 2 or 10 µg/mL U18666A. 450
Bar graphs represent the mean viral titers ± SEM of three independent experiments. One-way 451 ANOVA followed by by Dunnett's multiple comparison test. **p ≤ 0.01, ***p ≤ 0.001. 452 