key: cord-0700519-1akq1j4e
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: 2021-04-06
journal: Br J Pharmacol
DOI: 10.1111/bph.15418
sha: ca3c2e6a5254e48c01393b191a0001f935c22daa
doc_id: 700519
cord_uid: 1akq1j4e

BACKGROUND AND PURPOSE: The SARS‐COV‐2 pandemic and the global spread of coronavirus disease 2019 (COVID‐19) urgently call 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 interface of SARS CoV‐2 with the host via repurposing of clinically licensed drugs and evaluated their use in combinatory treatments with virus‐ and host‐directed drugs in vitro. EXPERIMENTAL APPROACH: We tested the antiviral potential of 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 the direct acting antiviral remdesivir, an inhibitor of viral RNA polymerase. KEY RESULTS: Drug treatments were well‐tolerated and potently impaired viral replication. Importantly, both itraconazole–remdesivir and fluoxetine–remdesivir combinations inhibited the production of infectious SARS‐CoV‐2 particles > 90% and displayed synergistic effects, as determined in commonly used reference models for drug interaction. CONCLUSION AND IMPLICATIONS: Itraconazole–remdesivir and fluoxetine–remdesivir combinations are promising starting points for therapeutic options to control SARS‐CoV‐2 infection and severe progression of COVID‐19.

The zoonotic coronavirus SARS-CoV-2 and the resulting coronavirus disease 2019 pandemic impressively show the global threat potential of a newly emerging zoonotic pathogen. More than 2 million people have died so far from the current outbreak and the proportion of infected people was estimated to reach more than 10% of the global population, with still unpredictable fatality rates (Baud et al., 2020; Rajgor et al., 2020; Wu et al., 2020) . Because of the pressing burden on national health systems and economic losses, safe and efficient treatment strategies are urgently required. While multiple candidate vaccines have been successfully developed in the last months, the rigorous testing and extensive clinical trials are timeconsuming processes and the production and distribution of effective and safe vaccines remain serious challenges. Thus, approaches other than immunization might offer useful additional options for the management and control of SARS-CoV-2 infection and the treatment of COVID-19 (Fierabracci et al., 2020) . A possibility to speed up the availability of drugs for the treatment of novel infections is the use of drugs that are already in clinical use for unrelated diseases via the so-called "drug repurposing." Because their safety profiles, dosages and side effects are already known, this approach represents a promising strategy to identify antiviral drugs with faster clinical implementation and lower development costs, considerations that are especially important in the global COVID-19 pandemic (Pushpakom et al., 2018) . In addition to drugs that directly target the virus, host cell components that are vitally important in the viral life cycle are explored as promising starting points for therapeutic intervention ("host cell-directed therapy") (Ianevski et al., 2020; Schwegmann & Brombacher, 2008; Zumla et al., 2020) .

Although proteolytic cleavage of the SARS-CoV-2 spike surface protein by the host cell transmembrane protease serine 2 (TMPRSS2) enables SARS-CoV-2 to directly fuse with the plasma membrane, endocytosed SARS-CoV-2 particles use endosomeresiding proteases for fusion within endosomes (Tang et al., 2020) .

Both pathways contribute to the SARS-CoV-2 infection process and the preferential use of the actual fusion pathway might critically depend on the presence of plasma membrane proteases (Hoffmann et al., 2020) . Our earlier research on influenza virus infection identified the late endosomal cholesterol balance as a critical factor in the influenza virus infection success and established this viral entry point as a possible pharmacological target. Elevated cholesterol levels inhibit the fusion of the influenza lipid envelope with the endosomal membranes and thus inhibit the efficient transfer of the viral genome into the host cytosol (Kühnl et al., 2018; Musiol et al., 2013) . We found that the clinically licensed antifungal itraconazole, a triazole derivative that blocks the fungal ergosterol pathway, has antiviral properties against a range of viruses and is effective against influenzas A virus infections in a preclinical mouse model (Schloer et al., 2019; . This additional therapeutic function is most likely based on direct inhibition of the endosomal cholesterol transporter Niemann-Pick Type C1 (NPC intracellular cholesterol transporter 1; NPC1; SLC65A1) and the subsequent cholesterol storage (Schloer et al., 2019; Trinh et al., 2017) .

The late endosome is an entry site for many zoonotically transmitted viruses, in particular for enveloped viruses including SARS-CoV-2 (Tang et al., 2020) . Because of the functional similarities in transmitting the viral genome into the host cell, the same endosomal components might serve as pharmacological targets for a broad host-directed antiviral strategy against such viruses. Continuing our work on the endosomal host-virus interface, we explored whether a similar repurposing strategy could be used to impair SARS-CoV-2 entry and infection. Therefore, we assessed clinically licensed drugs that also affect endolysosomal lipid storage and cholesterol build-up for their antiviral potential. Here, we report that itraconazole treatment potently inhibited the production of SARS-CoV-2 infectious particles. Together with our recently published work on the antiviral potential of the widely used 5-HT uptake inhibitor fluoxetine, which also negatively affects endosomal cholesterol release (Kornhuber et al., 2010; Schloer, Brunotte, et al., 2020) on SARS-CoV-2 infection, the results presented in this study strongly argue for the endolysosomal host-SARS-CoV-2 interface as a druggable target.

Combination therapy using several drugs that each target different molecular pathways is considered a key strategy to achieve therapeutic success with lower doses and a reduced likelihood of the development of drug resistance. Thus, we assessed the efficacy of a combined treatment using remdesivir, a nucleotide analogue prodrug that inhibits SARS-CoV-2 viral RNA-dependent RNA polymerase (Gordon et al., 2020) together with itraconazole or fluoxetine.

Both two-drug combinations showed stronger antiviral activities

• SARS-CoV-2 is susceptible to the broad-spectrum antiviral remdesivir.

• The antifungal itraconazole and the antidepressant fluoxetine can be repurposed to target enveloped viruses.

• Itraconazole, similar to fluoxetine, showed effective antiviral activity against SARS-CoV-2 infection in vitro,

• Combinatory treatments with itraconazole-remdesivir and fluoxetine-remdesivir displayed enhanced potency through synergy in vitro.

• Combinatory treatments of itraconazole and fluoxetine along remdesivir might offer superior treatment to inhibit SARS-CoV-2.

against SARS-CoV-2 compared to remdesivir monotherapy and pharmacodynamic evaluation via commonly used reference models to study drug interaction revealed synergistic interaction.

The human bronchial epithelial cell lines Calu-3 and the Vero E6 cells were cultivated in DMEM with 10% standardized FBS (FBS Superior; Merck), 2-mM L-glutamine, 100 UÁml −1 penicillin, 0.1 mgÁml −1 streptomycin and 1% non-essential amino acids (Merck) in a humidified incubator at 5% CO 2 and 37 C. Calu-3 monolayers were polarized and cultured as described . Itraconazole (2 mgÁml −1 , Sigma), fluoxetine (5 mM, Sigma) and remdesivir (10 mM, Hycultec) were solubilized in DMSO. The SARS-CoV-2 isolate hCoV-19/Germany/FI1103201/2020 (EPI-ISL_463008, mutation D614G in spike protein) was amplified on Vero E6 cells and used for the infection assays. 

To determine the number of infectious particles in the supernatant of treated cells, a standard plaque assay was performed. Briefly, 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 h at 37 C. The inoculum was replaced with 2× MEM (MEM containing 0.2% BSA, 2-mM L-glutamine, 1-M HEPES, pH 7.2, 7.5% NaHCO 3 , 100 UÁml −1 penicillin, 0.1 mgÁml −1 streptomycin and 0.4% Oxoid agar) and incubated at 37 C. Virus plaques were visualized by staining with neutral red and virus titres were calculated as plaque-forming units (PFU) per ml. , an open-source free stand-alone web application for the analysis of drug combination data (Ianevski et al., 2017) . Synergy was evaluated based on the zero interaction potency (ZIP), Bliss independence and highest single agent reference models (He et al., 2018) . We further analysed the overall drug combination sensitivity score by using the combination sensitivity score method (Malyutina et al., 2019) .

Itraconazole, Fluoxetine, DMSO, Staurosporine and MTT assay were obtained from Sigma, Remdesivir was purchased from Hyculture. 

Based on the successful repurposing of itraconazole for the treatment of influenza virus infection reported in our earlier studies (Schloer et al., 2019; , we first established whether this clinically licensed drug also had an antiviral potential on the production of infectious SARS-CoV-2 particles. In line with our previous results (Schloer, Brunotte, et al., 2020) , both Calu-3 and Vero (Schloer, Brunotte, et al., 2020) , we also assessed the effects of a combined fluoxetine/remdesivir (FluoRem) treatment in addition to the itraconazole/remdesivir (ItraRem) combination (Figure 2 ). Both drugs are clinically licensed and do not induce significant cytotoxicity over the whole concentration range (Schloer, Brunotte, et al., 2020; Figure S1a ). The combination treatments were also well tolerated and no cytotoxic effects were seen when cells were simultaneously treated with the drug pairs, thus excluding synergistic toxicity ( Figure S1b,c) .

For all drugs, we chose those concentrations that were not sufficient to achieve a 90% reduction when individually applied (Figure 3a ). For both ItraRem and FluoRem combinations, a potent reduction in virus titres was detected in all cases. Of note, several combinations yielded a reduction > 90% of the maximum virus titres produced in control cells (Figure 3b ). We next considered the pharmacological interactions of the respective drug pairs. Thus, we evaluated the drug interactions via Bliss independence, highest single agent and zero interaction potency, three commonly used reference synergy models that differ in their basic assumptions of drug interaction they are based on. The results, presented in Figures 4 and 5 , consistently argued for synergistic action of remdesivir with itraconazole and fluoxetine, as indicated by the positive average synergy score across all models. Closer inspection of the drug interaction relationships and landscape visualizations revealed that for ItraRem, the highest synergy scores were calculated with the lower concentration ranges of both drugs (Figure 4) . The strong synergy led to an overall drug combination sensitivity score of 89.64, resulting in >90% inhibition already at 500 nM of remdesivir F I G U R E 2 Antiviral activities of treatments. Infectious virus production in polarized Calu-3 cells treated with two-drug combinations as indicated 2 hours post-infection (hpi). Each symbol represents plaque-forming units (PFU) per ml detected in a single experimental sample; lines indicate means; n = 5 per treatment and 250 nM of itraconazole. FluoRem combination treatment had a higher average synergy score, as well as a higher combination sensitivity score than ItraRem (92.82 vs. 89.64), suggesting that this drug combination is more likely to show synergy. Importantly, for all models, the FluoRem combinations that met the ≥90% inhibition criterion were well within the high synergy area ( Figure 5 ).

To combat the current global health emergency caused by the SARS-CoV-2 pandemics, vaccines and antivirals are urgently needed. Entry of enveloped viruses such as SARS-CoV-2 into a host cell depends on the fusion of their lipid hull with the host membrane and the transfer F I G U R E 3 Antiviral activities of single and combination treatments. Polarized Calu-3 cells were treated with the indicated drug combinations for 48 h. (a) Single treatment; (b) combinatory treatment. Each symbol represents mean per cent inhibition ± SEM of infectious virus production, with mean virus titre produced in control cells (treated with the solvent DMSO) set to 100%; n = 5. Dotted line, 90% reduction in viral titre F I G U R E 4 Evaluation of the pharmacological interactions of itraconazole and remdesivir (ItraRem). zero interaction potency (ZIP), Bliss independence and highest single agent (HSA) reference models were used to assess the interaction landscapes and to identify areas of synergy. Interaction surfaces are colour coded according to the synergy scores of the responses F I G U R E 5 Evaluation of the pharmacological interactions of fluoxetine and remdesivir (FluoRem). zero interaction potency (ZIP), Bliss independence and highest single agent (HSA) reference models were used to assess the interaction landscapes and to identify areas of synergy. Interaction surfaces are colour coded according to the synergy scores of the responses of the viral genome into the cytosol. The SARS-CoV-2 spike protein, which protrudes from the virus surface, mediates initial binding to ACE2, which serves as the host cell surface receptor (Lan et al., 2020; Li et al., 2003; Ou et al., 2020; Zhou et al., 2020) . To promote fusion with the host cell membrane, the spike protein needs to be primed by proteolytic cleavage, which can be mediated by several host proteases. TMPRSS2-mediated cleavage leads to fusion with the plasma membrane, while endosome-residing proteases are utilized by endocytosed SARS-CoV-2 particles for fusion within endosomes. Since both routes have been reported to contribute to the SARS-CoV-2 infectivity (Hoffmann et al., 2020) , the endosomal compartment is also a critical host/pathogen interface for SARS-CoV-2. Our previous studies strongly support the late endosomal cholesterol balance as a cellular target for antiviral intervention (Kühnl et al., 2018; Musiol et al., 2013; Schloer et al., 2019; Schloer, Brunotte, et al., 2020; . Notably, our previous reports showed that endolysosomal lipid storage and cholesterol build-up could be induced via repurposing of drugs approved for unrelated applications.

We found that the triazole derivative itraconazole, a clinically licensed antifungal drug (World Health Organization, 2019) that directly inhibits the endosomal cholesterol transporter Niemann-Pick Type C1 (Trinh et al., 2017) , has an antiviral potential on the endosomal fusion of enveloped viruses including influenza viruses (Schloer et al., 2019) . Moreover, our previous study already revealed a beneficial antiviral activity in vivo (Schloer et al., 2019) .

The findings presented in this study add SARS-CoV-2 to the spectrum of itraconazole-sensitive enveloped viruses. Our results reveal a potent antiviral activity of itraconazole on the production of SARS-CoV-2 infectious particles, with EC 50 values comparable to what we previously reported for itraconazole-mediated antiviral activity against IAV subtypes (Schloer et al., 2019; .

The bioavailability after oral application of itraconazole is low because of the low water solubility of this highly lipophilic compound (Domínguez-Gil Hurlé et al., 2006; Grant Prentice & Glasmacher, 2005) . Because the slightly acidic salt is ionized only at very low pH, fluctuations in individual gastric acidity, for example fasting conditions or medications, reduce the bioavailability. Food or acidic beverages can influence the adsorption of orally administered itraconazole, depending on the formulation. Only limited amounts are absorbed from the gastrointestinal tract, leading to high interpatient variability in plasma levels (Allegra et al., 2017; Bae et al., 2011; Domínguez-Gil Hurlé et al., 2006; Lestner & Hope, 2013; Shin et al., 2004) . Yet itraconazole has been proven to be clinically useful in the prophylaxis and treatment of fungal infections, with a recommended daily dose of 200-400 mgÁday −1 , which might be increased up to 600 mgÁday −1 in case of severe infections. Effective plasma concentration ranges in patients with pulmonary aspergillosis treated with 200-mg itraconazole once daily have been reported to begin at 500 μgÁL −1 , well above the EC 50 levels for anti-SARS-CoV-2 antiviral activity determined in this study. We recently also discovered that the widely used antidepressant fluoxetine has strong SARS-CoV-2 antiviral activity (Schloer, Brunotte, et al., 2020) . In contrast to itraconazole, the bioavailability of orally administered fluoxetine is high, having plasma levels of 350 μgÁL −1 after 2 weeks and up to 1,055 μgÁL −1 after longer treatment periods were reported in patients on 20 mgÁday −1 fluoxetine (Pope & Zaraa, 2016; Preskorn et al., 1991) , levels that we found sufficient to inhibit over 50% of SARS-CoV-2 virus titres. Interestingly, fluoxetine is a racemic mixture of two enantiomers both of which have been reported to exert antiviral activity (Zimniak et al., 2020) , which might allow for the development of antiviral derivatives that do not affect serotonin re-uptake.

Together with the findings presented here on the inhibitory function of itraconazole treatment on SARS-CoV-2 replication, these results suggest that both drugs are promising candidates for repurposing as a host-directed drug for SARS-CoV-2 infection treatment. Both drugs most likely interfere with the proper endosomal cholesterol levels. Whereas itraconazole and posaconazole both directly inhibit the endosomal cholesterol transporter Niemann-Pick Type C1 (Trinh et al., 2017) , fluoxetine functionally blocks the endolysosome-residing enzyme sphingomyelin phosphodiesterase (acid sphingomyelinase), which in turn causes sphingomyelin accumulation and negatively affects cholesterol release from this compartment (Kornhuber et al., 2010) . However, host-directed drugs will rather cause impaired viral replication and suppress infection than completely eradicate the pathogen. The resulting demand for high drug doses and early and prolonged treatment is often associated with poor patient compliance.

While drugs directly acting on virus structures are much more likely to completely eliminate the pathogens in shorter treatment time, a major concern about virus-directed antivirals is the development of drug resistance. As observed with the influenza neuraminidase inhibitor oseltamivir (Kim et al., 2013) , viruses that evolve resistance mechanisms to evade the antiviral activity are increasingly emerging (Strasfeld & Chou, 2010) . Because profound changes would be required to allow viruses to replicate independently of otherwise essential host factors, combinatory treatments with both virus-and host-directed drugs are considered to overcome these shortcomings and are routinely explored for enhanced treatment success. The nucleoside analogue remdesivir, which was originally developed against Ebola (Warren et al., 2016) , is a direct acting antiviral that interferes with the viral RNA-dependent RNA polymerase. When remdesivir is incorporated into the viral RNA the synthesis is prematurely terminated and viral replication is inhibited (Gordon et al., 2020) . Remdesivir exerts antiviral activity against a range of viruses including Ebola, Marburg and MERS and is also active against SARS-CoV-2 (Agostini et al., 2018; Brown et al., 2019; Sheahan et al., 2017 Sheahan et al., , 2020 Warren et al., 2016) . Indeed, remdesivir was the first new drug to receive an FDA emergency use authorization for the treatment of severe COVID-19 cases, although its clinical use is still controversially. A major obstacle is the very short half-life, with initial peak serum concentrations of about 3 mgÁL −1 measured directly after intravenous infusion rapidly declining to 80-170 μgÁL −1 after 1 h (Tempestilli et al., 2020 ). Yet recently published results of doubleblind, randomized, placebo-controlled trials suggest that remdesivir treatment is beneficial in the treatment of hospitalized COVID-19 patients and might be improved further by combination therapy with remdesivir and the host-directed Janus kinase inhibitor baricitinib Kalil et al., 2020) . Interestingly, GS-441524, the predominant remdesivir metabolite in the plasma which has a considerably longer half-life, has been reported to potently inhibit SARS-CoV-2 replication in vitro as well as in a mouse model of SARS-CoV-2 infection and pathogenesis , making this metabolite a promising drug candidate.

Here, we report that ItraRem and FluoRem drug combinations, in both cases targeting the host cell and the virus independently, showed stronger antiviral activities against SARS-CoV-2 than the remdesivir monotherapy. Moreover, the overall therapeutic effect of the combinations was larger than the expected sum of the independent drug effects and underlying synergistic effects were determined, allowing for lower concentrations of the individual drugs. Of note, their reported plasma concentrations are well within these ranges.

While our results support the use of the direct antiviral remdesivir in combination with a host-directed drug as a potential therapeutic strategy to reduce SARS-CoV-2 infection success and to limit deleterious COVID-19 outcomes, the translation of in vitro results into the clinics remains a major challenge. Although pharmacokinetic and safety profiles for these drugs are available, combined medications bear the risk of drug-drug interactions which may lead to reduced therapeutic benefit or even severe adverse effects. Therefore, it is important to mention that a vast number of drugs are currently known to interact with itraconazole, fluoxetine and even remdesivir (information available at Drugs.com 2021). Furthermore, the patient's genetic disposition and physiological or pathophysiological conditions demand for a careful evaluation of the appropriate treatment strategy.

Thus, it is paramount to evaluate the patient-related risks and benefits, as coadministration of drugs might be contraindicated in certain patients.

Our analysis on the antiviral activity of combinatory drug combinations via commonly used interaction models argues for an enhanced efficacy that is based on synergistic drug interaction and suggests promising novel options for SARS-CoV-2 treatment.

Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease

The Concise Guide to PHAR-MACOLOGY

The Concise Guide to PHARMA-COLOGY

Evaluation of posaconazole pharmacokinetics in adult patients with invasive fungal infection

Increased oral bioavailability of itraconazole and its active metabolite, 7-hydroxyitraconazole, when coadministered with a vitamin C beverage in healthy participants

Real estimates of mortality following COVID-19 infection. The Lancet Infectious Diseases

Remdesivir for the treatment of Covid-19-Final report

Broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent RNA dependent RNA polymerase

Experimental design and analysis and their reporting II: Updated and simplified guidance for authors and peer reviewers

Therapeutic drug monitoring of itraconazole and the relevance of pharmacokinetic interactions

G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences

COVID-19: A review on diagnosis, treatment, and prophylaxis

Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency

Making sense of itraconazole pharmacokinetics

Methods for high-throughput drug combination screening and synergy scoring

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

SynergyFinder: A web application for analyzing drug combination dose-response matrix data

Potential antiviral options against SARS-CoV-2 infection

Baricitinib plus remdesivir for hospitalized adults with Covid-19

Mechanism-based covalent neuraminidase inhibitors with broad-spectrum influenza antiviral activity

Functional inhibitors of acid sphingomyelinase (FIASMAs): A novel pharmacological group of drugs with broad clinical applications

Late endosomal/lysosomal cholesterol accumulation is a host cell-protective mechanism inhibiting endosomal release of influenza A virus

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Itraconazole: An update on pharmacology and clinical use for treatment of invasive and allergic fungal infections

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus

Remdesivir metabolite GS-441524 effectively inhibits SARS-CoV-2 infection in mice models

Drug combination sensitivity scoring facilitates the discovery of synergistic and efficacious drug combinations in cancer

Annexin A6-balanced late endosomal cholesterol controls influenza a replication and propagation

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

Serum fluoxetine and norfluoxetine levels support the safety of fluoxetine in overdose

Antidepressant response and plasma concentrations of fluoxetine

Remdesivir inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice

Drug repurposing: Progress, challenges and recommendations

The many estimates of the COVID-19 case fatality rate. The Lancet Infectious Diseases

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

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

Combinatory treatment with oseltamivir and itraconazole targeting both virus and host factors in influenza a virus infection

Host-directed drug targeting of factors hijacked by pathogens

Broadspectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses

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

Dose-dependent pharmacokinetics of itraconazole after intravenous or oral administration to rats: Intestinal first-pass effect. Antimicrobial Agents and Chemotherapy

Antiviral drug resistance: Mechanisms and clinical implications

Coronavirus membrane fusion mechanism offers a potential target for antiviral development

Pharmacokinetics of remdesivir and GS-441524 in two critically ill patients who recovered from COVID-19

Triazoles inhibit cholesterol export from lysosomes by binding to NPC1

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

World Health Organization model list of essential medicines (Geneve)

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

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

The serotonin reuptake inhibitor fluoxetine inhibits SARS-CoV-2

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

Drug synergy of combinatory treatment with remdesivir and the repurposed drugs fluoxetine and itraconazole effectively impairs SARS-CoV-2 infection in vitro

We thank Jonathan Hentrey for help with the plaque assays. 

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. S.S., S.L. and U.R. are members of the German 

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

https://orcid.org/0000-0001-8074-7400Jing Tang https://orcid.org/0000-0001-7480-7710Stephan Ludwig https://orcid.org/0000-0003-4490-3052Ursula Rescher https://orcid.org/0000-0001-8892-319X