Marion Plaze

David Attali

Matthieu Prot

Anne-Cécile Petit

Michael Blatzer

Fabien Vinckier

Laurine Levillayer

Jeanne Chiaravalli

Florent Perin-Dureau

Arnaud Cachia

Gérard Friedlander

Fabrice Chrétien

Etienne Simon-Loriere

Raphaël Gaillard
email: r.gaillard@ghu-paris.fr

Inhibition of the replication of SARS-CoV-2 in human cells by the FDA-approved drug chlorpromazine

Keywords: chlorpromazine, SARS-CoV-2, COVID-19, repurposing of molecules, human cells Chemical compounds studied

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Introduction

From the beginning of the COVID-19 outbreak we observed in Sainte Anne hospital (GHU PARIS Psychiatrie & Neurosciences, Paris, France) a higher prevalence of symptomatic and severe forms of COVID-19 infections among health care professionals (~14%) compared to patients in psychiatric wards (~4%) [START_REF] Plaze | Repositionnement de la chlorpromazine dans le traitement du COVID-19: étude reCoVery[END_REF]. This unexpected finding, that patients with more comorbidities and risk factors (overweight, cardiovascular disorders, etc.) seem to be protected against symptomatic and severe forms of COVID-19, drew our interest to decipher putative factors that could mediate this anti-SARS-CoV-2 protection. Because patients in psychiatric wards receive psychotropic medications, we screened the literature for antiviral effects associated with those drugs. This literature analysis identified chlorpromazine (CPZ), the prototype of phenothiazine-type antipsychotics, as the lead candidate [START_REF] Plaze | Repositionnement de la chlorpromazine dans le traitement du COVID-19: étude reCoVery[END_REF]. The reasons why CPZ was identified as the lead candidate among psychotropic drugs are multiple. Firstly, in addition to its antipsychotic activity, CPZ has been used for decades in virology. On the one hand, in vitro studies have demonstrated CPZ antiviral properties, for example against influenza [START_REF] Krizanová | Influence of chlorpromazine on the replication of influenza virus in chick embryo cells[END_REF],

hepatitis viruses [START_REF] Blanchard | Hepatitis C virus entry depends on clathrin-mediated endocytosis[END_REF], alphaviruses [START_REF] Pohjala | Inhibitors of Alphavirus Entry and Replication Identified with a Stable Chikungunya Replicon Cell Line and Virus-Based Assays[END_REF], JC virus [START_REF] Pho | JC virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis[END_REF], Japanese encephalitis virus [START_REF] Nawa | Interference in Japanese encephalitis virus infection of Vero cells by a cationic amphiphilic drug, chlorpromazine[END_REF], bronchitis-virus [START_REF] Chu | Avian infectious bronchitis virus enters cells via the endocytic pathway[END_REF], MHV-2 [START_REF] Pu | Mouse Hepatitis Virus Type 2 Enters Cells through a Clathrin-Mediated Endocytic Pathway Independent of Eps15[END_REF], Zika virus [START_REF] Persaud | Infection by Zika viruses requires the transmembrane protein AXL, endocytosis and low pH[END_REF] or dengue virus [START_REF] Carro | Blockade of dengue virus entry into myeloid cells by endocytic inhibitors in the presence or absence of antibodies[END_REF]. On the other hand, CPZ is the leading drug inhibiting clathrin-mediated endocytosis [START_REF] Daniel | Phenothiazine-derived antipsychotic drugs inhibit dynamin and clathrin-mediated endocytosis[END_REF][START_REF] Wang | Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation[END_REF][START_REF] Chen | Clathrin-mediated endocytosis is a candidate entry sorting mechanism for Bombyx mori cypovirus[END_REF][START_REF] Inoue | Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic tail deleted[END_REF] -via translocation of clathrin and AP2 from the cell surface to intracellular endosomes [START_REF] Wang | Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation[END_REF] -and therefore is commonly used to determine the pathways of entry of viruses into cells [START_REF] Daniel | Phenothiazine-derived antipsychotic drugs inhibit dynamin and clathrin-mediated endocytosis[END_REF][START_REF] Wang | Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation[END_REF][START_REF] Chen | Clathrin-mediated endocytosis is a candidate entry sorting mechanism for Bombyx mori cypovirus[END_REF][START_REF] Inoue | Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic tail deleted[END_REF]. In a recent review article, the authors underlined the therapeutic potential of targeting clathrin-mediated endocytosis -essential for coronavirus cell entry [START_REF] Burkard | Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner[END_REF] -to tackle SARS-CoV-2 [START_REF] Yang | Targeting the Endocytic Pathway and Autophagy Process as a Novel Therapeutic Strategy in COVID-19[END_REF].

Secondly, CPZ has been shown to have antiviral activity against coronaviruses in multiple studies [START_REF] De Wilde | Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture[END_REF][START_REF] Dyall | Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection[END_REF][START_REF] Cong | MERS-CoV pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells[END_REF][START_REF] Weston | Broad anti-coronaviral activity of FDA approved drugs against SARS-CoV-2 in vitro and SARS-CoV in vivo[END_REF]. It was identified active against both MERS-CoV and SARS-CoV-1 in a screen of 348 FDA-approved drugs, together with three other compounds (chloroquine, loperamide, lopinavir), using different cell lines [START_REF] De Wilde | Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture[END_REF]. Similar results were obtained in a different library screen [START_REF] Dyall | Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection[END_REF], as well as in another study using primary human monocytes [START_REF] Cong | MERS-CoV pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells[END_REF]. More recently, CPZ (as well as 16 other compounds) has been shown to have antiviral activity against SARS-CoV-2 on monkey Vero E6 cells [START_REF] Weston | Broad anti-coronaviral activity of FDA approved drugs against SARS-CoV-2 in vitro and SARS-CoV in vivo[END_REF].

In this context, the aim of the current study was to investigate in vitro CPZ antiviral activity against SARS-CoV-2 on monkey Vero E6 cells as well as -for the first time -on human alveolar basal epithelial cells.

Material and methods

Cell culture and virus isolates

Vero E6 cells (African green monkey kidney epithelial cells, ATCC, CRL-1586) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS) and 5 units/mL penicillin and 5 μg/mL streptomycin at 37°C with 5% CO2. A549-ACE2 cells (adenocarcinomic human alveolar basal epithelial cells, transduced to express the human Angiotensin-converting enzyme 2 (ACE2), kind gift of Pr. O. Schwartz, Institut Pasteur, Paris France) were maintained in DMEM containing 10% FBS, 5 units/mL penicillin and 5 μg/mL streptomycin and 40 µg/mL blasticidin at 37°C with 5% CO2.

SARS-CoV

Antiviral activity assay

Cells were seeded into 96-well plates 24 h prior to the experiment. Two hours prior to infection, cell culture supernatant was replaced with media containing 32µM, 16µM, 8µM, 4µM and 2µM of CPZ, or the equivalent volume of maximum H2O vehicle used as a control. For the infection, the drug-containing media was removed, and replaced with virus inoculum (MOI of 0.1 PFU/cell for VeroE6 and 1 for A549-ACE2) for 2 hours. The inoculum was then removed and replaced with 100µl fresh media (2% FBS) containing CPZ at the indicated concentrations or H2O and incubated for 48 hours. At 48h, cell supernatant was collected and spun for 5 min at 3,000g to remove debris. Toxicity controls were setup in parallel on uninfected cells. RNA was extracted from 50µl aliquots of supernatant using the Nucleospin 96 virus kit (Macherey-Nagel) following the manufacturer's instructions. Detection of viral genomes was performed by RT-qPCR, using the IP4 primer set developed by the NRC at Institut Pasteur (described on the WHO website (https://www.who.int/docs/default-source/coronaviruse/real-time-rt-pcrassays-for-the-detection-of-sars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2). RT-qPCR was performed using the Luna Universal One-Step RT-qPCR Kit (NEB) in an Applied Biosystems QuantStudio 3 thermocycler, using the following cycling conditions: 55°C for 10 min, 95°C for 1 min, and 40 cycles of 95°C for 10 sec, followed by 60°C for 1 min. The quantity of viral genomes is expressed as PFU equivalents, and was calculated by performing a standard curve with RNA derived from a viral stock with a known viral titre.

Cell viability in drug-treated cells was measured using the AlamarBlue reagent (ThermoFisher). At 48 h post treatment, the drug-containing media was removed and replaced with AlamarBlue and incubated for 2h at 37°C. Fluorescence was measured in a Tecan Infinity 2000 plate reader.

Using the same experimental setting, we tested the antiviral activity of remdesivir, as a comparator and a validator of the experiment.

Data analysis

Antiviral activity was assayed as a percentage of inhibition of SARS-CoV-2, normalized to the quantity of viral genomes under the lowest concentration of CPZ (i.e. 2 µM) for each of the three independent replicates.

Percentage cytotoxicity was calculated relative to untreated cells (0% toxicity). Antiviral activity and cytoxicity data were analysed using GraphPad Prism version 8.4.2 for MacOS (GraphPad Software, San Diego, California USA, www.graphpad.com). Non-linear regressions were performed and IC50 (half maximal inhibitory concentration), IC90 (90% maximal inhibitory concentration), CC50 (half maximal cytotoxic concentration) and SI (selectivity index, calculated by dividing the CC50 by the IC50) were calculated from "[Agonist] vs. response --Variable slope" curves with constraints to remain above 0% and below 100%.

Results

African Green Monkey VeroE6 cells and human alveolar basal epithelial A549-ACE2 cells were infected with SARS-CoV-2 in presence of different concentrations of CPZ. Supernatants were harvested at day 2 and analysed by RT-qPCR for the presence of SARS-CoV-2 RNA. In parallel, cell viability was assessed on noninfected cells.

In monkey VeroE6 cells, we measured an antiviral activity of CPZ against SARS-CoV-2, with an IC50 of 8.2 µM, an IC90 of 15.2 µM, a CC50 of 13.5µM and thus a SI of 1.65 (Figure 1.A).

In human A549-ACE2 cells, CPZ was also associated with an anti-SARS-CoV-2 activity, with an IC50 of 11.3 µM (Figure 1.B) and an IC90 of 14.3 µM. CPZ was associated with a cytotoxic effect in this model at the highest doses assessed, with a CC50 of 23.1 µM and therefore a SI of 2.04 (Figure 1.B).

Remdesivir was associated with an antiviral activity against SARS-CoV-2, with an IC50 of 5 µM in monkey VeroE6 cells, and an IC50 of 0.15 µM in human A549-ACE2 cells.

Figure 1. Antiviral activity of CPZ against SARS-CoV-2 in vitro in monkey VeroE6 cells (A) and human A549-ACE2 cells (B). Viral load in supernatants were measured at 48h (left Y axis), and viability under increasing concentrations of the antiviral compound are shown. Error bars denote s.e.m.

Discussion

With more than 6 000 000 infections and 370 000 deaths worldwide in just a few months [START_REF]COVID-19 situation update worldwide[END_REF], tools are urgently needed to help against the SARS-CoV-2 pandemic, to diminish disease severity along with contagiousness and to reduce the socio-economic consequences of the pandemic. In this study, we evidenced in vitro antiviral activity of CPZ against a SARS-CoV-2 isolate in monkey and human cells, with IC50 of respectively 8.2 and 11.3 μM. These results are in line with previous demonstrations of antiviral properties of CPZ, a well-known inhibitor of clathrin-mediated endocytosis [START_REF] Daniel | Phenothiazine-derived antipsychotic drugs inhibit dynamin and clathrin-mediated endocytosis[END_REF][START_REF] Wang | Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation[END_REF][START_REF] Chen | Clathrin-mediated endocytosis is a candidate entry sorting mechanism for Bombyx mori cypovirus[END_REF][START_REF] Inoue | Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic tail deleted[END_REF], against older coronaviruses. Besides, the measured IC50 of remdesivir are consistent with previously published work [START_REF] Wang | Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro[END_REF][START_REF] Jeon | Identification of Antiviral Drug Candidates against SARS-CoV-2 from FDA-Approved Drugs[END_REF], reinforcing the validation of our experimental setup.

Cytotoxicity (%) Antiviral activity Cytotoxicity

A B

Even though the measured SI of CPZ are very low, such IC50 measured in vitro may translate to CPZ dosage used in clinical routine. Indeed, one of the main advantages of using CPZ against SARS-CoV-2 could lie in its biodistribution (Figure 2), and mainly and foremost in its pneumophilic properties. In 1968, Forrest et al.

quantified the CPZ distribution in selected organs through a post-mortem study of 6 patients with schizophrenia and treated by CPZ until their death [START_REF] Forrest | Distribution of chlorpromazine metabolites in selected organs of psychiatric patients chronically dosed up to the time of death[END_REF]. Among the 5 patients with available lung measurements, the highest CPZ concentration was found in the lungs. These high pneumophilic properties of CPZ have also been described in preclinical studies, reporting CPZ concentration in the lungs 20 to 200 times higher than in plasma after a single dose of CPZ [START_REF] Forrest | Distribution of chlorpromazine metabolites in selected organs of psychiatric patients chronically dosed up to the time of death[END_REF][START_REF] Bickel | Distribution of chlorpromazine and imipramine in adipose and other tissues of rats[END_REF][START_REF] Fujii | Autoradiographic and biochemical studies of drug distribution in the liver. II. [35S]Chlorpromazine and [14C]imipramine[END_REF][START_REF] Sgaragli | Rat tissue concentrations of chlorimipramine, chlorpromazine and their N-demethylated metabolites after a single oral dose of the parent compounds[END_REF]. Besides the lungs, CPZ concentrations have been demonstrated to be very high In the salivary glands, 30-100 times higher than in plasma after a single dose of CPZ [START_REF] Fujii | Autoradiographic and biochemical studies of drug distribution in the liver. II. [35S]Chlorpromazine and [14C]imipramine[END_REF][START_REF] May | Chlorpromazine levels and the outcome of treatment in schizophrenic patients[END_REF]. In humans, May et al. studied saliva concentration of CPZ in 48 newly admitted patients with schizophrenia, and saliva concentrations were found to be between 1300 to 22000 ng/ml (i.e. 4.1 to 69 μM;

CPZ molar mass = 318.86 g/mol) 1 to 8 hours after a single dose of CPZ [START_REF] May | Chlorpromazine levels and the outcome of treatment in schizophrenic patients[END_REF]. These high saliva concentrations of CPZ could reduce the contagiousness of COVID-19 [START_REF] To | Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study[END_REF]. Moreover, because of its lipophilic nature, CPZ cross the blood-brain barrier [START_REF] Rundle-Thiele | Repurposing some older drugs that cross the blood-brain barrier and have potential anticancer activity to provide new treatment options for glioblastoma[END_REF]. CPZ brain distribution, underlying its antipsychotic action and side effects, has been described with brain to plasma concentration ratio of up to 50 in rodents [START_REF] Bickel | Distribution of chlorpromazine and imipramine in adipose and other tissues of rats[END_REF][START_REF] Fujii | Autoradiographic and biochemical studies of drug distribution in the liver. II. [35S]Chlorpromazine and [14C]imipramine[END_REF][START_REF] Sgaragli | Rat tissue concentrations of chlorimipramine, chlorpromazine and their N-demethylated metabolites after a single oral dose of the parent compounds[END_REF][START_REF] Wiesel | The distribution and metabolism of chlorpromazine in rats and the relationship to effects on cerebral monoamine metabolism[END_REF]. In humans, CPZ distribution in the brain was studied in 22 patients with schizophrenia in our hospital in 1979, with brain to plasma ratio ranging from 15 to 25 [START_REF] Comar | Brain distribution and kinetics of 11C-chlorpromazine in schizophrenics: positron emission tomography studies[END_REF]. This could be of great interest for treating or preventing the neurological and psychiatric forms of COVID-19 [START_REF] Rogers | Psychiatric and neuropsychiatric presentations associated with severe coronavirus infections: a systematic review and meta-analysis with comparison to the COVID-19 pandemic[END_REF] with, to date, no available therapeutic options. Indeed, remdesivir, anti-IL6 drug tocilizumab and hydroxychloroquine, three of the most studied drugs in the treatment of COVID-19, do not cross, or cross to a far lesser extent the blood-brain barrier [START_REF] Warren | Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys[END_REF][START_REF] Acharya | Management of cytokine release syndrome and neurotoxicity in chimeric antigen receptor (CAR) T cell therapy[END_REF][START_REF] Hunter | CAR T-cell associated neurotoxicity: Mechanisms, clinicopathologic correlates, and future directions[END_REF][START_REF] Richardson | CNS penetration of potential anti-COVID-19 drugs[END_REF]. Overall, even though the extrapolation from in vitro to clinically relevant dosage is not straightforward, the IC50 of 11.3 μM (i.e. 3603 ng/ml) measured in vitro in human cells may be compatible with CPZ dosage used in clinical routine. Indeed, residual plasma levels of CPZ in patients range from 30 to 300 ng/ml [START_REF] Hiemke | Consensus Guidelines for Therapeutic Drug Monitoring in Neuropsychopharmacology: Update 2017[END_REF], which could correspond to 600 -60 000 ng/ml in lungs [START_REF] Forrest | Distribution of chlorpromazine metabolites in selected organs of psychiatric patients chronically dosed up to the time of death[END_REF][START_REF] Bickel | Distribution of chlorpromazine and imipramine in adipose and other tissues of rats[END_REF][START_REF] Fujii | Autoradiographic and biochemical studies of drug distribution in the liver. II. [35S]Chlorpromazine and [14C]imipramine[END_REF][START_REF] Sgaragli | Rat tissue concentrations of chlorimipramine, chlorpromazine and their N-demethylated metabolites after a single oral dose of the parent compounds[END_REF] and 900 -30 000 ng/ml in saliva [START_REF] Fujii | Autoradiographic and biochemical studies of drug distribution in the liver. II. [35S]Chlorpromazine and [14C]imipramine[END_REF][START_REF] May | Chlorpromazine levels and the outcome of treatment in schizophrenic patients[END_REF] according to our review (Figure 2). This extrapolation is supported by our observation of lower prevalence of symptomatic and severe forms of COVID-19 infections in psychiatric patients.

Repurposing CPZ, a molecule already used in clinical practice, could offer both ready-to-use treatment with well-known and very mild side effects. CPZ has been widely used in clinical routine in the treatment of acute and chronic psychoses for decades. This first antipsychotic medication was discovered in 1952 by Jean Delay and Pierre Deniker at Sainte Anne hospital [START_REF] Delay | Therapeutic use in psychiatry of phenothiazine of central elective action (4560 RP)][END_REF]. At this time, CPZ is prescribed for around 70 years and FDAapproved in psychiatry and anaesthesiology, with an excellent tolerance profile. CPZ is also used in refractory nausea and vomiting of pregnancy [START_REF]Committee on Practice Bulletins-Obstetrics. ACOG Practice Bulletin No. 189: Nausea And Vomiting Of Pregnancy[END_REF], in advanced cancer [START_REF] Gupta | Nausea and Vomiting in Advanced Cancer-The Cleveland Clinic Protocol (TH310)[END_REF], and to treat refractory headaches in various neurological conditions [START_REF] Marmura | The Acute Treatment of Migraine in Adults: The American Headache Society Evidence Assessment of Migraine Pharmacotherapies[END_REF].

Conclusions

This first in vitro study of CPZ antiviral activity against SARS-CoV-2 in monkey and human cells supports that CPZ, a well-known drug with antiviral properties and an excellent tolerance profile, could be tested clinically as an alternative to currently used drugs or combinations of drugs in COVID-19 treatment. This proof of principle for the feasibility of CPZ as anti-SARS-CoV-2 therapeutic is a critical step for future clinical trial (NCT04366739).

  -2, isolate BetaCoV/France/IDF0372/2020 C2, was supplied by the National Reference Centre for Respiratory Viruses (NRC) hosted at Institut Pasteur (Paris, France) and headed by Pr. S. Van der Werf. The human sample from which this strain was isolated has been provided by Dr. X. Lescure and Pr. Y. Yazdanpanah from the Bichat Hospital, Paris, France. Viral stocks were prepared by propagation in VeroE6 cells in DMEM supplemented with 2% FBS. Viral titres were determined by plaque assay. All experiments involving live SARS-CoV-2 were performed in compliance with Institut Pasteur guidelines for Biosafety Level 3 work. All experiments were performed in at least three biologically independent replicates.

Figure 2 .

 2 Figure 2. Review of temporal CPZ biodistribution in lung, saliva and brain. Ratio of tissue to plasma CPZ

Acknowledgments

We are grateful to the Centre National de Reference des virus des infections respiratoires for sharing reagents and protocols. We thank Olivier Schwartz and his team for sharing the A549-ACE2 cell line. The authors wish to thank the Fondation Pierre Deniker for its support.