key: cord-270329-t60t639i
authors: Schloer, Sebastian; Brunotte, Linda; Mecate-Zambrano, Angeles; Zheng, Shuyu; Tang, Jing; Ludwig, Stephan; Rescher, Ursula
title: Drug synergy of combinatory treatment with remdesivir and the repurposed drugs fluoxetine and itraconazole effectively impairs SARS-CoV-2 infection in vitro
date: 2020-10-16
journal: bioRxiv
DOI: 10.1101/2020.10.16.342410
sha: 
doc_id: 270329
cord_uid: t60t639i

The SARS-COV-2 pandemic and the global spread of coronavirus disease 2019 (COVID-19) urgently calls for efficient and safe antiviral treatment strategies. A straightforward approach to speed up drug development at lower costs is drug repurposing. Here we investigated the therapeutic potential of targeting the host- SARS-CoV-2 interface via repurposing of clinically licensed drugs and evaluated their use in combinatory treatments with virus- and host-directed drugs. We tested the antiviral potential of repurposing the antifungal itraconazole and the antidepressant fluoxetine on the production of infectious SARS-CoV-2 particles in the polarized Calu-3 cell culture model and evaluated the added benefit of a combinatory use of these host-directed drugs with remdesivir, an inhibitor of viral RNA polymerase. Drug treatments were well-tolerated and potent impaired viral replication was observed with all drug treatments. Importantly, both itraconazole-remdesivir and fluoxetine-remdesivir combinations inhibited the production of infectious SARS-CoV-2 particles > 90% and displayed synergistic effects in commonly used reference models for drug interaction. Itraconazole-Remdesivir and Fluoxetine-Remdesivir combinations are promising therapeutic options to control SARS-CoV-2 infection and severe progression of COVID-19.

The zoonotic coronavirus SARS-CoV-2 and the resulting COVID-19 pandemic impressively 

The late endosome is an entry site for many zoonotically transmitted viruses, in particular for 

the results presented in this study strongly argue for the endolysosomal host-SARs-CoV-2 84 interface as a druggable target. However, host-directed drugs will rather suppress infection 85 than completely eradicate the pathogen. The resulting demand for high drug doses and 86 early and prolonged treatment is often associated with poor patient compliance. While drugs 87 directly acting on virus structures are much more likely to completely eliminate the 88 pathogens in shorter treatment time, emerging viral resistance to these antivirals is a major 89 concern, as observed with the influenza neuraminidase inhibitor oseltamivir (Kim et al., itraconazole antiviral activity in SARS-CoV-2 infected Vero cells (Fig. 1b) . Of note, no 172 detectable cytotoxicity was observed with these doses (Fig. S1a) . The itraconazole cytotoxic 197 S1a). The combination treatments were also well-tolerated, and no cytotoxic effects were 198 seen when cells were simultaneously treated with the drug pairs (Fig. S1b, c) . For all drugs, we chose those concentrations that were not sufficient to achieve a 90% 202 reduction when individually applied (Fig 3a) . For both ItraRem and FluoRem combinations, a 203 potent reduction in virus titers was detected in all cases. Of note, several combinations 204 yielded a reduction > 90% of the maximum virus titers produced in control cells (Fig. 3b) . synergy scores were calculated with the lower concentration ranges of both drugs (Fig. 4) .

The strong synergy led to an overall drug combination sensitivity score (CSS) of 89.64, 224 resulting in >90% inhibition already at 500 nM of remdesivir and 250 nM of itraconazole.

FluoRem combination treatment had a higher average synergy score, as well as a higher 226 CSS score than ItraRem (92.82 vs 89.64), suggesting that this drug combination is more 227 likely to show synergy. Importantly, for all models, the FluoRem combinations that met the ≥ 228 90% inhibition criterion were well within the high synergy area (Fig. 5) . 

While remdesivir and the host-directed drugs itraconazole or fluoxetine target independent 297 pathways, we found that drug combinations together with remdesivir (ItraRem and 298 FluoRem) showed stronger antiviral activities against SARS-CoV-2 than the remdesivir 299 monotherapy. Moreover, the overall therapeutic effect of the combinations was larger than 

SARS-CoV-2 on virus entry and its immune cross-reactivity with 395 SARS-CoV

Remdesivir Inhibits SARS-CoV-2 in Human Lung Cells and Chimeric 398 SARS-CoV Expressing the SARS-CoV-2 RNA Polymerase in Mice

Drug 401 repurposing: Progress, challenges and recommendations

The many 404 estimates of the COVID-19 case fatality rate

Targeting the endolysosomal host-SARS-CoV-2 interface by clinically licensed 407 functional inhibitors of acid sphingomyelinase (FIASMA) including the antidepressant 408 fluoxetine

The 410 clinically licensed antifungal drug itraconazole inhibits influenza virus in vitro and in vivo

Combinatory 413 Treatment with Oseltamivir and Itraconazole Targeting Both Virus and Host Factors in 414 Influenza A Virus Infection

Host-directed drug targeting of factors 416 hijacked by pathogens

Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic 419 coronaviruses

Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and 422 interferon beta against MERS-CoV

Dose-Dependent Pharmacokinetics 424 of Itraconazole after Intravenous or Oral Administration to Rats: Intestinal First-Pass 425

Antiviral drug resistance: Mechanisms and clinical 427 implications

Coronavirus 429 membrane fusion mechanism offers a potential target for antiviral development

Triazoles 432 inhibit cholesterol export from lysosomes by binding to NPC1

Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus 436 monkeys

Estimating clinical severity of COVID-19 from the transmission dynamics in Wuhan

A 441 pneumonia outbreak associated with a new coronavirus of probable bat origin

Reducing mortality 444 from 2019-nCoV: host-directed therapies should be an option

SUPPORTING FIGURE 1 Analysis of the cytotoxicity of treatments. (a) Itraconazole, (b)

Calu-3 cells were treated with the 449 indicated drug combinations for 48 h. Bars display mean percentages of viable 450 cells ± SEM, with mean viability in solvent-treated control cells (C) set to 100%

Staurosporine (ST)-induced cytotoxicity served as a positive control. n = 5, one-way 452 ANOVA followed by Dunnett's multiple comparison test

SUPPORTING FIGURE 2 Analysis Dose-Response curve of remdesivir treatments in

Calu-3 cells were infected with 0.1 MOI of SARS-CoV-2 for 1 h and treated with 456 the indicated drug combinations for 48 h. Mean percent inhibition ± SEM of SARS-CoV-2 457 replication, with mean virus titer in control cells (treated with the solvent DMSO) set to 100%

LogEC 50 and LogEC 90 values were determined by fitting a non-linear regression 459 model

We thank Jonathan Hentrey for help with the plaque assays.

This research was funded by grants from the German Research Foundation (DFG), 308 CRC1009 "Breaking Barriers", Project A06 (to U.R.) and B02 (to S.L.), CRC 1348 "Dynamic