ORIGINAL RESEARCH published: 25 March 2021 doi: 10.3389/fphar.2021.646676 Identification of Plitidepsin as Potent Inhibitor of SARS-CoV-2-Induced Cytopathic Effect After a Drug Repurposing Screen Edited by: Jordi Rodon 1, Jordana Muñoz-Basagoiti 2†, Daniel Perez-Zsolt 2†, Marc Noguera-Julian 2,3, Maria Cristina Albertini, Roger Paredes 2,4, Lourdes Mateu 4, Carles Quiñones 4, Carles Perez 5, Itziar Erkizia 2, University of Urbino Carlo Bo, Italy Ignacio Blanco 6, Alfonso Valencia 5,7, Víctor Guallar 5,7, Jorge Carrillo 2, Julià Blanco 2,3,6, Reviewed by: Joaquim Segalés 8,9, Bonaventura Clotet 2,3,6, Júlia Vergara-Alert 1* and Arnab K. Chatterjee, Nuria Izquierdo-Useros 2,6* Calibr at Scripps Research, 1 United States IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la UAB, Bellaterra, Spain, 2IrsiCaixa AIDS Research Stephen J. Polyak, Institute, Badalona, Spain, 3University of Vic–Central University of Catalonia (UVic-UCC), Vic, Spain, 4Germans Trias i Pujol University of Washington, Hospital, Badalona, Spain, 5Barcelona Supercomputing Center, Barcelona, Spain, 6Germans Trias i Pujol Research Institute United States (IGTP), Can Ruti Campus, Badalona, Spain, 7Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain, 8 *Correspondence: UAB, CReSA (IRTA-UAB), Campus de la UAB, Bellaterra, Spain, 9Departament de Sanitat i Anatomia Animals, Facultat de Júlia Vergara-Alert Veterinària, Bellaterra, Spain julia.vergara@irta.cat Nuria Izquierdo-Useros There is an urgent need to identify therapeutics for the treatment of Coronavirus disease nizquierdo@irsicaixa.es † 2019 (COVID-19). Although different antivirals are given for the clinical management of These authors have contributed equally to this work SARS-CoV-2 infection, their efficacy is still under evaluation. Here, we have screened existing drugs approved for human use in a variety of diseases, to compare how they Specialty section: counteract SARS-CoV-2-induced cytopathic effect and viral replication in vitro. Among the This article was submitted to Respiratory Pharmacology, potential 72 antivirals tested herein that were previously proposed to inhibit SARS-CoV-2 a section of the journal infection, only 18 % had an IC50 below 25 µM or 102 IU/ml. These included plitidepsin, Frontiers in Pharmacology novel cathepsin inhibitors, nelfinavir mesylate hydrate, interferon 2-alpha, interferon- Received: 27 December 2020 gamma, fenofibrate, camostat along the well-known remdesivir and chloroquine Accepted: 01 February 2021 Published: 25 March 2021 derivatives. Plitidepsin was the only clinically approved drug displaying nanomolar Citation: efficacy. Four of these families, including novel cathepsin inhibitors, blocked viral entry Rodon J, Muñoz-Basagoiti J, in a cell—type specific manner. Since the most effective antivirals usually combine Perez-Zsolt D, Noguera-Julian M, Paredes R, Mateu L, Quiñones C, therapies that tackle the virus at different steps of infection, we also assessed several Perez C, Erkizia I, Blanco I, Valencia A, drug combinations. Although no particular synergy was found, inhibitory combinations did Guallar V, Carrillo J, Blanco J, not reduce their antiviral activity. Thus, these combinations could decrease the potential Segalés J, Clotet B, Vergara-Alert J and Izquierdo-Useros N (2021) emergence of resistant viruses. Antivirals prioritized herein identify novel compounds and Identification of Plitidepsin as Potent their mode of action, while independently replicating the activity of a reduced proportion of Inhibitor of SARS-CoV-2-Induced Cytopathic Effect After a Drug drugs which are mostly approved for clinical use. Combinations of these drugs should be Repurposing Screen. tested in animal models to inform the design of fast track clinical trials. Front. Pharmacol. 12:646676. doi: 10.3389/fphar.2021.646676 Keywords: SARS-CoV-2, antivirals, plitidepsin, synergy, viral entry Frontiers in Pharmacology | www.frontiersin.org 1 March 2021 | Volume 12 | Article 646676 Rodon et al. Plitidepsin potently inhibits SARS-CoV-2 INTRODUCTION then bud into the lumen of endoplasmic reticulum (ER)-Golgi compartments, where viruses are finally released into the A novel betacoronavirus, the severe acute respiratory syndrome extracellular space by exocytosis. Potentially, any of these viral coronavirus 2 (SARS-CoV-2), is causing a respiratory disease cycle steps could be targeted with antivirals, so we have thus pandemic that began in Wuhan, China, in November 2019, and searched for these compounds as well. has now spread across the world (Chen et al., 2020b). To date, Finally, as the most effective antiviral treatments are usually remdesivir is the only approved antiviral drug for the specific based on combined therapies that tackle distinct steps of the viral treatment of this coronavirus infectious disease 2019 or COVID- life cycle, we also tested the active compounds in combination. 19 (Beigel et al., 2020; Grein et al., 2020). However, several drugs These combinations may be critical to abrogate the potential are being used in the frontline of clinical management of SARS- emergence of resistant viruses and to increase antiviral activity, CoV-2-infected individuals in hospitals all around the world, to enhancing the chances to improve clinical outcome. try to avoid the development of the COVID-19 associated pneumonia, which can be fatal. By the end of December 2020, almost 1.7 million people had died from COVID-19, and over 75 MATERIAL AND METHODS million cases have been reported (https://covid19.who.int). Although different drug regimens are being applied to Cell Cultures hospitalized patients, no clinical study has evidenced their Vero E6 cells (ATCC CRL-1586) were cultured in Dulbecco’s efficacy yet. Under this scenario, initiatives launched by the modified Eagle medium (DMEM; Lonza) supplemented with 5% World Health Organization (WHO), such as the fetal calf serum (FCS; EuroClone), 100 U/ml penicillin, 100 μg/ml SOLIDARITY study that has compared remdesivir, streptomycin, and 2 mM glutamine (all ThermoFisher Scientific). hydroxychloroquine, ritonavir/lopinavir and ritonavir/lopinavir HEK-293T (ATCC repository) were maintained in DMEM with plus ß-interferon regimes, have been of critical importance to 10% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml prioritize the use of the most active compounds (WHO, 2020). streptomycin (all from Invitrogen). HEK-293T overexpressing Unfortunately, although remdesivir has proven efficacy in the human ACE2 were kindly provided by Integral Molecular randomized controlled trials (Beigel et al., 2020; Grein et al., Company and maintained in DMEM (Invitrogen) with 10% fetal 2020), a recent update of the WHO clinical trial has failed to bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin, detect any effect on overall mortality, initiation of ventilation and and 1 μg/ml of puromycin (all from Invitrogen). TMPRSS2 duration of hospital stay with any of the antivirals tested (Pan, human plasmid (Origene) was transfected using 2020). Thus, there is an urgent need to identify novel therapeutic X-tremeGENE HP Transfection Reagent (Merck) on HEK- approaches for individuals with COVID-19 developing severe 293T overexpressing the human ACE2 and maintained in the disease and fatal outcomes. previously described media containing 1 mg/ml of geneticin In this report we present a prioritized list of effective (Invitrogen) to obtain TMPRSS2/ACE2 HEK-293T cells. compounds with proven antiviral efficacy in vitro to halt SARS-CoV-2 replication. Compounds were analyzed Virus isolation, Titration and Sequencing depending on their expected mechanism of action, to identify SARS-CoV-2 was isolated from a nasopharyngeal swab candidates tackling diverse steps of the viral life cycle. SARS- collected from an 89-year-old male patient giving informed CoV-2 entry requires viral binding and spike protein activation consent and treated with Betaferon and hydroxychloroquine for via interaction with the cellular receptor ACE2 and the cellular 2 days before sample collection. The swab was collected in 3 ml protease TMPRSS2 at the plasma membrane (Hoffmann et al., medium (Deltaswab VICUM) to reduce viscosity and stored at 2020). Interference with either of these ligands has proven to −80°C until use. Vero E6 cells were cultured on a cell culture decrease SARS-CoV-2 infectivity (Monteil et al. 2020; Hoffmann flask (25 cm2) at 1.5 x 106 cells overnight prior to inoculation et al., 2020), and therefore, inhibitors targeting viral entry may with 1 ml of the processed sample, for 1 h at 37°C and 5% CO2. prove valuable. In addition, SARS-CoV-2 enters into the cells via Afterward, 4 ml of 2% FCS-supplemented DMEM were supplied endocytosis and accumulates in endosomes where cellular and cells were incubated for 48 h. Supernatant was harvested, cathepsins can also prime the spike protein and favor viral centrifuged at 200 × g for 10 min to remove cell debris and fusion upon cleavage (Simmons et al., 2005; Mingo et al., stored at −80°C. Cells were assessed daily for cytopathic effect 2015; Hoffmann et al., 2020), providing additional targets for and the supernatant was subjected to viral RNA extraction and antiviral activity. Once SARS-CoV-2 fuses with cellular specific RT-qPCR using the SARS-CoV-2 UpE, RdRp and N membranes, it triggers viral RNA release into the cytoplasm, assays (Corman et al., 2020). The virus was propagated for two where polyproteins are translated and cleaved by proteases (Song passages and a virus stock was prepared collecting the et al., 2019). This leads to the formation of an RNA supernatant from Vero E6. replicase—transcriptase complex driving the production of Viral RNA was extracted directly from the virus stock using several negative—stranded RNA via both replication and the Indimag Pathogen kit (Indical Biosciences) and transcribed to transcription (Song et al., 2019). Numerous negative-stranded RNAs transcribe into messenger RNA genomes, allowing for the ™ cDNA using the PrimeScript RT reagent Kit (Takara) using oligo-dT and random hexamers, according to the manufacturer’s translation of viral nucleoproteins, which assemble in viral protocol. DNA library preparation was performed using SWIFT capsids at the cytoplasm (Song et al., 2019). These capsids amplicon SARS-CoV-2 panel (Swift Biosciences). Sequencing Frontiers in Pharmacology | www.frontiersin.org 2 March 2021 | Volume 12 | Article 646676 Rodon et al. Plitidepsin potently inhibits SARS-CoV-2 ready libraries where then loaded onto Illumina MiSeq platform measured in a Fluoroskan Ascent FL luminometer (ThermoFisher and a 300 bp paired-end sequencing kit. Sequence reads were Scientific). quality filtered and adapter primer sequences were trimmed using trimmomatic. Amplification primer sequences were removed IC50 Calculation and Statistical Analysis using cutadapt (Martin, 2011). Sequencing reads were then Response curves of compounds or their mixes were adjusted to a mapped against coronavirus reference (NC_045512.2) using non-linear fit regression model, calculated with a four-parameter bowtie2 tool (Langmead and Salzberg, 2012). Consensus logistic curve with variable slope. Cells not exposed to the virus genomic sequence was called from the resulting alignment at a were used as negative controls of infection, and were set as 100% 18 × 1800 × 879 average coverage using samtools (Li et al., 2009). of viability to normalize data and calculate the percentage of Genomic sequence was deposited at GISAID repository (http:// cytopathic effect. Statistical differences from 100% were assessed gisaid.org) with accession ID EPI_ISL_510689. with a one sample t test. All analyses and figures were generated with the GraphPad Prism v8.0b Software. Pseudovirus Production HIV-1 reporter pseudoviruses expressing SARS-CoV-2 Spike In Silico Drug Modeling protein and luciferase were generated using two plasmids. pNL4- We performed Glide docking on SARS-CoV-2 3CL Main 3. Luc.R-E-was obtained from the NIH AIDS repository. SARS- protease using the DrugBank database (https://go.drugbank. CoV-2. SctΔ19 was generated (Geneart) from the full protein com), with a total of 6837 molecules containing FDA approved sequence of SARS-CoV-2 spike with a deletion of the last 19 drugs, reported phased I/II/III drugs and experimental amino acids in C-terminal, human-codon optimized and molecules (i.e. drugs that are at the preclinical or animal inserted into pcDNA3.4-TOPO (Ou et al., 2020b). Spike plasmid testing stage) (Gupta and Zhou, 2020). For this, two was transfected with X-tremeGENE HP Transfection Reagent different receptors were prepared, the 6LU7 pdb structure, (Merck) into HEK-293T cells, and 24 h post-transfection, cells after removing the covalently bound inhibitor, and a were transfected with pNL4-3. Luc.R-.E-. Supernatants were combination of two crystals from the Diamond collection harvested 48 h later, filtered with 0.45 µM (Millex Millipore) and (https://www.diamond.ac.uk/covid-19). This combination stored at −80°C until use. Control pseudoviruses were obtained by intended to open a bit more the active site by rotating replacing the spike plasmid by a VSV-G plasmid (kindly provided Gln189 and exposing it more to the solvent, after visual by Dr Andrea Cimarelli). The p24gag content of all viruses was inspection of the different fragment crystals. Receptors were quantified using an ELISA (Perkin Elmer) and viruses were titrated prepared with the Schrodinger’s protein wizard assigning in HEK-293T overexpressing the human ACE2. protonation states at pH 7 using the interactive optimizer. Also, after inspection of the recent crystal structures, Glide Antivirals and Compounds SP docking was performed with two different (ligand) The complete list of compounds used for this study and vendors hydrogen bond constraints: Glu16 and His163 (this last are shown in Supplementary Tables S1–S5. Drugs were used at residue with epsilon protonation); we enforced single concentrations ranging from 100 μM to 0.0512 nM at 5-fold serial constraints and also attempted the combination of both. The dilutions. NPOs were used at concentrations ranging from 10 μM best 11 molecules, based on Glides’s docking score, were to 0.00512 nM at 5-fold serial dilutions. Plitidepsin was also selected for experimental validation. Notice, however, that assayed at concentrations ranging from 10 μM to 0.5 nM at 3- top docking scores, did not exceed −9, indicating poor fold dilutions. Interferons were assayed at concentrations ranging potential binding, in agreement with other non-productive from 104 to 0.0005 IU/ml at 5-fold serial dilutions. When two repurposing efforts reported on SARS-CoV-2 main protease, drugs were combined, each one was added at a 1:1 molar ratio at including sophisticated in silico studies (Gupta and Zhou, concentrations ranging from 100 μM to 0.0512 nM at 5-fold serial 2020). dilutions. In combination with other drugs, plitidepsin was also assayed at concentrations ranging from 10 μM to 0.5 nM at 3-fold Pseudovirus Assay dilutions. HEK-293T overexpressing the human ACE2 and TMPRSS2 were used to test antivirals at the concentrations found to be Antiviral Activity effective for SARS-CoV-2 without toxicity, which were the Increasing concentrations of antiviral compounds were added to following: 5 µM for niclosamide; 10 µM for chloroquine, Vero E6 cells and immediately after, we added 101.8 TCID50/mL chlorpromazine, ciclesonide, MDL 28170 and fenofibrate; of SARS-CoV-2, a concentration that achieves a 50% of cytopathic 20 µM for hydroxychloroquine, CA-074-Me and arbidol HCl; effect (20 TCID50 per well to 30,000 cells in 200 µl). Untreated 25 µM for E-64d; 50 µM for Baricitinib; 100 µM for non-infected cells and untreated virus-infected cells were used as Amantadine, NB-DNJ, 3’ sialyl-lactose Na salt, tofacitinib, negative and positive controls of infection, respectively. To detect and Camostat mesylate; 1000 µM for methyl-β-cyclodextrin, any drug-associated cytotoxic effect, Vero E6 cells were equally and 12,5 mg/ml for AAT. A constant pseudoviral titer was cultured in the presence of increasing drug concentrations, but in used to pulse cells in the presence of the drugs. At 48 h post- the absence of virus. Cytopathic or cytotoxic effects of the virus or inoculation, cells were lysed with the Glo Luciferase system drugs were measured 3 days after infection, using the CellTiter- (Promega). Luminescence was measured with an EnSight Glo luminescent cell viability assay (Promega). Luminescence was Multimode Plate Reader (Perkin Elmer). Frontiers in Pharmacology | www.frontiersin.org 3 March 2021 | Volume 12 | Article 646676 Rodon et al. Plitidepsin potently inhibits SARS-CoV-2 FIGURE 1 | Antiviral activity of entry inhibitors against SARS-CoV-2. (A) Antiviral activity of hydroxychloroquine and azithromycin. Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of hydroxychloroquine, azithromycin, and their combination. Drugs were used at a concentration ranging from 0.0512 nM to 100 µM. When combined, each drug was added at the same concentration. Non-linear fit to a variable response curve from one representative experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. The particular IC50 value of this graph is indicated. Cytotoxic effect on Vero E6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). (B) Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of amantadine, a clathrin-mediated (Continued ) Frontiers in Pharmacology | www.frontiersin.org 4 March 2021 | Volume 12 | Article 646676 Rodon et al. Plitidepsin potently inhibits SARS-CoV-2 FIGURE 1 | endocytosis inhibitor, E-64d, a pan-cathepsin inhibitor acting downstream once viruses are internalized in endosomes, NB-DNJ, an inhibitor of ganglioside biosynthesis and methyl-β-cyclodextrin, a cholesterol-depleting agent. All drugs were used at a concentration ranging from 0.0512 nM to 100 µM aside from methyl- β-cyclodextrin, which was used 10 times more concentrated. Non-linear fit to a variable response curve from one experiment with two replicates is shown (red lines). Cytotoxic effect on Vero E6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). (C) Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of camostat, a TMPRSS2 inhibitor, and ATT, an alpha-1 antyitrypsin, a broad cellular protease inhibitor, as described in (A). (D) Effect of entry inhibitors on luciferase expression of reporter lentiviruses pseudotyped with SARS-CoV-2 Spike in ACE2 expressing HEK-293T cells. Values are normalized to luciferase expression by mock-treated cells set at 100%. Mean and s.e.m. from two experiments with one to three replicates. Cells were exposed to fixed amounts of SARS-CoV-2 Spike lentiviruses in the presence of a non-toxic constant concentration of the drugs tested on Vero E6. Significant statistical deviations from 100% were assessed with a one sample t test. (E) Comparison of entry inhibitors blocking viral endocytosis, such as chloroquine, with inhibitors blocking serine protease TMPRSS2 expressed on the cellular membrane, such as camostat, on different cell lines. ACE2 expressing HEK- 293T cells transfected or not with TMPRSS2 were exposed to SARS-CoV-2 Spike lentiviruses as described in (B). Values are normalized to luciferase expression by mock-treated cells set at 100%. Mean and s.e.m. from at least two representative experiments with two replicates. Statistical deviations from 100% were assessed with a one sample t test. SARS-CoV-2 Detection in the Supernatant against SARS-CoV-2-induced cellular cytotoxicity on Vero E6 of Infected Cells cells (Liu et al., 2020). As shown in Figure 1A, this drug was able Viral accumulation in the supernatant of Vero E6 cells infected as to inhibit viral-induced cytopathic effects at concentrations where described previously in the presence of increasing concentrations no cytotoxic effects of the drug were observed, as previously of the indicated antiviral compounds was measured at day 3 post- reported (Liu et al., 2020; Wang et al., 2020). Since infection. The amount of SARS-CoV-2 nucleoprotein released to hydroxychloroquine was first administered in combination the supernatant was measured with an ELISA (SinoBiologicals), with the antibiotic azithromycin (Gautret et al., 2020), which according to the manufacturer’s protocol. This ELISA contains a induces antiviral responses in bronchial epithelial cells (Gielen buffer to lyse viral membranes and release nucleoprotein content. et al., 2010), we tested this antibiotic alone and in combination (Figure 1A), but found a similar activity to that of the chloroquine derivative alone (Figure 1A). Indeed, this was RESULTS also the case when we tested hydroxychloroquine in combination with different HIV-1 protease inhibitors and We have tested the antiviral activity of different clinically other relevant compounds currently being tested in clinical available compounds and their combinations by assessing their trials (Supplementary Table S2). ability to inhibit viral induced cytopathic effect in vitro. Our Additional Food and Drug Administration (FDA)-approved strategy was circumscribed mostly to compounds approved for compounds previously used to abrogate viral entry via clathrin- clinical use, since they are ideal candidates for entering into fast mediated endocytosis such as amantadine or chlorpromazine track clinical trials. Drug selection criteria first focused on were also tested in this SARS-CoV-2-induced cytotoxicity assay compounds already being tested in clinical trials, along with (Supplementary Table S2). Yet, we did not find any prominent well-known human immunodeficiency virus-1 (HIV-1) and effect of these agents; only a partial inhibition at 100 μM for hepatitis C virus (HCV) inhibitors, as well as other amantadine (Figure 1B). The broad cathepsin B/L inhibitor E64- compounds suggested to have potential activity against SARS- d also showed partial inhibitory activity (Figure 1B). E64-d exerts CoV-2 in molecular docking analysis or in vitro assays (Chen activity against viruses cleaved by cellular cathepsins upon et al., 2020a; Dittmar et al., 2020; Ianevski et al., 2020; Riva et al., endosomal internalization, as previously described using 2020; Weston et al., 2020). pseudotyped SARS-CoV-2 (Hoffmann et al., 2020). While We first assessed the activity of 16 compounds with these results could not be confirmed using the specific hypothetical capacity to inhibit viral entry, and then we cathepsin B inhibitor CA-074-Me due to drug-associated focused on 22 drugs thought to block viral replication upon toxicity (Supplementary Table S1), none of these cathepsin SARS-CoV-2 fusion. Molecular docking studies provided an inhibitors is approved for clinical use. It has also been additional 11 candidates, which were predicted to inhibit the suggested that hydroxychloroquine could block SARS-CoV-2 SARS-CoV-2 main protease. Finally, 23 compounds with spike interaction with GM1 gangliosides (Fantini et al., 2020). unknown mechanism of action were also assessed. By these GM1 gangliosides are enriched in cholesterol domains of the means, we have compared 72 drugs and 28 of their plasma membrane and have been previously shown to bind to combinations for their capacity to counteract SARS-CoV-2- SARS-CoV spike protein (Lu et al., 2008). This mode of viral induced cytopathic effect in vitro. interaction is aligned with the capacity of methyl-beta cyclodextrin, which depletes cholesterol from the plasma membrane to abrogate SARS-CoV-2 induced cytopathic effect Antiviral Activity of Compounds that (Figure 1B), as previously reported for SARS-CoV (Lu et al., Potentially Inhibit Viral Entry 2008). Removal of cholesterol redirected ACE2 receptor to other We first tested compounds that could have an effect before viral domains, but did not alter the expression of the viral receptor (Lu entry by impairing virus-cell fusion (Supplementary Table S1). et al., 2008). Moreover, NB-DNJ, an inhibitor of ganglioside We confirmed the inhibitory effect of hydroxychloroquine biosynthesis pathway, also decreased SARS-CoV-2 cytopathic Frontiers in Pharmacology | www.frontiersin.org 5 March 2021 | Volume 12 | Article 646676 Rodon et al. Plitidepsin potently inhibits SARS-CoV-2 effect (Figure 1B). These results highlight the possible role of highlight that alternative routes govern SARS-CoV-2 viral gangliosides in viral binding, although in soluble competition the entry and these pathways vary depending on the cellular polar head group of GM3 ganglioside (3’ Sialyllactose) was not target. Thus, effective treatments may need to block both able to reduce viral-induced cytopathic effect (Supplementary plasma membrane fusion and endosomal routes to fully Table S1). Of note, previously reported antiviral agents such as achieve viral suppression. the autophagy BECN1-stabilizing compounds niclosamide or ciclesonide (Gassen et al., 2020), (Jeon et al., 2020), the membrane lipid intercalating compound arbidol (Haviernik Antiviral activity of Compounds that et al., 2018) and the two JAK inhibitors baricitinib and Potentially Inhibit Post-entry Steps tofacitinib (Richardson et al., 2020; Stebbing et al., 2020) failed In our search for antivirals inhibiting post-viral entry steps, we to protected Vero E6 cells from SARS-CoV-2 induced cytopathic first focused on remdesivir, which has in vitro activity against effect (Supplementary Table S1). We next tested camostat, a SARS-CoV-2 after viral entry (Wang et al., 2020) and has already serine protease inhibitor with capacity to abrogate SARS-CoV-2 been approved for the treatment of COVID-19 by the FDA and Spike priming on the plasma membrane of human pulmonary the European Medicines Agency (EMA). We further confirmed cells and avoid viral fusion (Hoffmann et al., 2020). Camostat the in vitro capacity of remdesivir to inhibit SARS-CoV-2- showed no antiviral effect on Vero E6 cells (Figure 1C), what induced cytopathic effect on Vero E6 (Figure 2A). The IC50 indicates that the alternative viral endocytic route is the most value of this drug in repeated experiments was always below prominent entry route in this renal cell type. A broader cellular 10 µM (Supplementary Table S2). In combination with protease inhibitor such as alpha-1 antitrypsin (AAT), used to increasing concentrations of hydroxychloroquine, however, treat severe AAT human deficiency and currently under clinical remdesivir did not significantly modified its own antiviral study for COVID-19 due to its anti-inflammatory potential, has effect (Figure 2B). This was also the case for other antivirals already shown capacity to limit SARS-CoV-2 entry in cells tested in combination (Supplementary Table S3). Of note, other expressing TMPRSS2 (Münch, 2020; Oguntuyo et al., 2020). RNA polymerase inhibitors such as galdesivir, which was When we tested AAT antiviral efficacy on Vero E6, AAT proposed to tightly bind to SARS-CoV-2 RNA-dependent exerted an effect but required high concentrations that will RNA polymerase (Elfiky, 2020), showed no antiviral effect most likely rely on the activity of these proteases in the (Supplementary Table S3). Favipiravir, approved by the endosomal route (Figure 1C). National Medical Products Administration of China as the To confirm that identified compounds listed in first anti-COVID-19 drug in China (Tu et al., 2020), showed Supplementary Table S1 specifically inhibit the viral entry only partial inhibitory activity at the non-toxic concentration of step, we employed a luciferase-based assay using 100 µM (Supplementary Table S3). pseudotyped lentivirus expressing the spike protein of We also assessed clinically approved protease inhibitors with SARS-CoV-2, which allows to detect viral fusion on HEK- potent activity against HIV-1. However, none of the HIV-1 293 T cells transfected with ACE2. As a control, we used protease inhibitors detailed in Supplementary Table S3 lentiviruses pseudotyped with a VSV glycoprotein, where no showed remarkable protective antiviral activity against SARS- entry inhibition above 20 % was detected for any of the drugs CoV-2 infection on Vero E6 cells, with the exception of tested (data not shown). In sharp contrast, SARS-CoV-2 nelfinavir mesylate hydrate, which showed an IC50 value pseudoviruses were effectively blocked by most of the drugs below 10 µM (Supplementary Table S3 and Figure 2C). previously tested on Vero E6 with wild-type virus (Figure 1D). Lopinavir and tipranavir inhibited SARS-CoV-2-induced The main differences were observed with CA-074-Me, cytopathic effect at the non-toxic concentration of 20 μM, and ciclesonide and arbidol. These compounds showed a partial amprenavir exhibited activity at the non-toxic concentration of blocking effect on ACE2 HEK-293T cells that was not obvious 100 µM (Figure 2C). Darunavir, which has been tested in clinical when using replication competent SARS-CoV-2 on Vero E6 trials, showed partial inhibitory activity at 100 μM, although this (Supplementary Figure S1). In addition, NB-DNJ failed to concentration had 8.5 ± 6.2% of cytotoxicity associated block viral entry (Figure 1D). Overall, using alternative SARS- (Figure 2C). Of note, we tested HIV-1 reverse transcriptase CoV-2 viral systems, we could identify chloroquine inhibitors such as tenofovir disoproxil fumarate, emtricitabin, derivatives, cathepsin inhibitors and cholesterol depleting tenofovir alafenamide, and their combinations, but they also agents as the most promising candidates to block SARS- failed to show any antiviral effect against SARS-CoV-2 CoV-2 endocytosis in Vero E6 and HEK-293T cells (Supplementary Table S1). These results indicate that future transfected with ACE2. However, chloroquine derivatives clinical trials should contemplate the limited antiviral effect were the only ones that displayed an IC50 below 25 µM displayed by these anti-HIV-1 inhibitors against SARS-CoV- (Table 1), and were also active abrogating pseudoviral entry 2 in vitro. into HEK-293 T cells expressing ACE2 (Figure 1E). Although We also assessed the inhibitory capacity of HCV inhibitors, camostat failed to inhibit viral fusion on ACE2 HEK-293T cells but none showed any antiviral activity (Supplementary Table (Figure 1E), its activity was rescued when these cells were S3). Of note, exogenous interferon-2α and interferon-γ displayed transfected with TMPRSS2. The opposite effect was observed antiviral activity against SARS-CoV-2 (Supplementary Table for chloroquine, which reduced its inhibitory activity on S3). In light of these results, we tested the inhibitory effect of TMPRSS2 transfected cells (Figure 1E). Our results the TLR 7 agonist vesatolimod that triggers interferon Frontiers in Pharmacology | www.frontiersin.org 6 March 2021 | Volume 12 | Article 646676 Rodon et al. Plitidepsin potently inhibits SARS-CoV-2 TABLE 1 | Compounds with antiviral activity ordered depending on their IC50 values on Vero E6. Notice that plitidepsin and cathepsin inhibitors display IC50 values in the nM range, while the rest of the compounds are in the µM or 102 IU/ml range. Drug IC50/CC50 μM Mode of action Previous clinical Vendor Origin (Mean +/− SD) use nM Plitidepsin 0.06 +/− 0.02/> 0.1 Targets eukaryotic Elongation Factor 1A2 Multiple myeloma PharmaMar (eEF1A2) MDL 28170 0.14 +/− 0.06/> 87 Calpain III and Cathepsin B inhibitor Pre-clinical Merck NPO-2142; -2143 ∼0.54/>10 Calpain and Cathepsin Inhibitors Pre-clinical Landsteiner and-2260 Genmed Remdesivir 2.16 +/− 4.1/> 85 RNA Polymerase inhibitor Ebola virus Cayman Chemical Hydroxycloroquine 10.9 +/− 11.3/> 96 Clathrin-mediated endocitosys or pH- Malaria Laboratories dependent viral fusion inhibitor Rubio <25 μM or Nelfinavir mesylate Not calculated, but active < 10/ Protease inhibitor HIV-1 Sigma Aldrich 102IU/mL hydrate > 25 Chloroquine 3.86/>25 Clathrin-mediated endocitosys or pH- Malaria Sigma Aldrich dependent viral fusion inhibitor Interferon-2α 8.1+/−0.7 × 102 IU/mL/> 100 IFN stimulated antivirus proteins Hepatitis and HIV-1 Sigma Aldrich × 102 IU/mL interferon-γ 11.2 × 102 IU/mL/> 100 × IFN stimulated antivirus proteins Granulomatous Sigma Aldrich 102 IU/mL disease Fenofibrate 19.8 +/− 8/>100 Activates PPARα Dyslipidemia Lacer production. Although this agonist was not able to protect from infection (Supplementary Table S5). Such was the case of the viral-induced cytopathic effect on Vero E6 (Supplementary ivermectin, an FDA-approved broad spectrum anti-parasitic Table S3), as expected since it is an interferon-producer deficient agent previously reported to inhibit the replication of SARS- cell line (Emeny and Morgan, 1979), it could still be useful in CoV-2 in vitro as measured by RNA accumulation (Caly et al., other competent cellular targets. Since severe COVID-19 patients 2020). display impaired interferon responses (Hadjadj et al., 2020), these However, among these potential antivirals, only three types strategies may be valuable to avoid disease complication. In of molecules exerted detectable antiviral activity in our assay: addition, we also assessed several compounds with the best itraconazole, fenofibrate, and calpain and cathepsin inhibitors computational docking scores among approved drugs against such as MDL 28170 and NPO compounds. Itraconazole, an the 3CL protease of SARS-CoV-2, but none of them were effective antifungal that may interfere with internal SARS-CoV-2 to protect Vero E6 from viral induced cytopathic effect budding within infected cells (Wu et al., 2020), displayed an (Supplementary Table S4). IC50 value of 80 µM (Figure 3A and Supplementary Table S5). The most potent antiviral tested was plitidepsin (Figure 2D), Fenofibrate is clinically used to treat dyslipidemia via activation which targets the eukaryotic Elongation Factor 1A2 (eEF1A2) of PPARα, and also inhibited the cytopathic effect exerted by and has been previously used for the treatment of multiple SARS-CoV-2 on Vero E6 at 20 µM (Figure 3B and myeloma. The mean IC50 value of this drug in repeated Supplementary Table S5). As fenofibrate is a regulator of experiments was always in nM concentrations cellular lipid metabolism, we made use of the luciferase- (Supplementary Table S3). In combination with other active based viral entry assay to try to elucidate its mode of action. antivirals, we did not observe a reduction on IC50 values When lentiviruses pseudotyped with the spike protein of SARS- (Supplementary Table S2). This result indicates no significant CoV-2 were added to ACE2-expressing HEK-293 T cells in the synergy, but also highlights the possibility of using plitidepsin presence of fenofibrate, viral entry was abrogated (Figure 3C). without reducing its antiviral activity in combined therapies The most potent agent found was MDL 28170, a calpain III (Figure 2D), what could be relevant to avoid possible selection inhibitor in a pre-clinical stage of development that displayed of resistant viruses. Overall, plitidepsin showed the lowest IC50 activity in the nanomolar range (Figure 3D and values of all the compounds tested in this in vitro screening Supplementary Table S5), as previously identified in vitro (Table 1). (Riva et al., 2020). Moreover, three out of four different calpain and cathepsin inhibitors named NPO showed potent antiviral activity too (Supplementary Figure S2). Of note, in combination with other active antivirals, we did not observe a Antiviral Activity of Compounds with reduction on IC50 values of MDL 28170 (Supplementary Unknown Mechanism of Action Table S2). We also assessed the inhibitory capacity of several inhibitors and Inhibitors of calpains, which are cysteine proteases, might broad anti-bacterial, anti-parasitic, anti-malarial, anti-influenza impair the activity of viral proteases like 3CL (main protease) and anti-fungal compounds, along with other pharmacological and PLpro (papain-like protease) (Schneider et al., 2012; Riva agents previously suggested to interfere with SARS-CoV-2 et al., 2020). However, calpain inhibitors may also inhibit Frontiers in Pharmacology | www.frontiersin.org 7 March 2021 | Volume 12 | Article 646676 Rodon et al. Plitidepsin potently inhibits SARS-CoV-2 FIGURE 2 | Antiviral activity of post-entry inhibitors. (A) Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of Remdesivir. Drug was used at a concentration ranging from 0.0512 nM to 100 μM. Non-linear fit to a variable response curve from one representative experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. The particular IC50 value of this graph is indicated. Cytotoxic effect on Vero E6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). (B). Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of remdesivir and its combination with hydroxychloroquine, as detailed in (A). Drugs in combination were used at a concentration ranging from 0.0512 nM to 100 μM (left panel). (C). Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of protease inhibitors against HIV-1. Nelfinavir mesylate hydrate was the only drug with activity. Inhibitors were used at a concentration ranging from 0.0512 nM to 100 μM. The particular IC50 value of this graph is indicated (D). Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of plitidepsin and its combinations with hydroxychloroquine and remdesivir. When combined, each drug was added at the same concentration. Drugs were used at a concentration ranging from 0.5 nM to 10 μM. The particular IC50 value of these graphs is indicated. cathepsin B-mediated processing of viral spike proteins or In conclusion, among the 72 compounds and their 28 glycoproteins, including SARS-CoV and Ebola (Schneider combinations tested herein for their potential capacity to abrogate et al., 2012; Zhou and Simmons, 2012). To understand the SARS-CoV-2 cytopathic effect, we only found 13 compounds with mechanisms of action of calpain and cathepsin inhibitors antiviral activity including camostat, and only eight types of these such as MDL 28170, we added lentiviruses pseudotyped with drugs had an IC50 below 25 µM or 102 IU/ml (Table 1). These eight the spike protein of SARS-CoV-2 to ACE2-expressing HEK- families of compounds were able to abrogate SARS-CoV-2 release to 293T cells and the same cells also expressing TMPRSS2 in the the supernatant in a dose dependent manner (Figure 4), indicating presence of this drug. Importantly, MDL 28170 only blocked that the reduction in the cytopathic effect that we had measured in viral entry in ACE2-expressing cells (Figure 3E). This result cells correlates with viral production. In the case of fenofibrate, indicates that MDL 28170 blocks cathepsins that are however, this compound was less active inhibiting viral release as implicated in SARS-CoV-2 entry via the alternative compared to the rest of the inhibitors tested (Figure 4), while still endosomal pathway, as described for chloroquine preserved cells from viral induced -cytopathic effect. As the eight derivatives and E-64d (Figure 3E), which are all active families of compounds found tackle different steps of the viral life when TMPRSS2 is not present and their inhibitor camostat cycle, they could be tested in combined therapies to abrogate the displays no activity (Figure 3E). potential emergence of resistant viruses. Frontiers in Pharmacology | www.frontiersin.org 8 March 2021 | Volume 12 | Article 646676 Rodon et al. Plitidepsin potently inhibits SARS-CoV-2 FIGURE 3 | Antiviral activity of inhibitors with unknown mechanism of action. (A). Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of Itraconazole. Drug was used at a concentration ranging from 0.0512 nM to 100 µ. Non-linear fit to a variable response curve from one representative experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. The particular IC50 value of this graph is indicated. Cytotoxic effect on Vero E6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). (B). Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of Fenofibrate, as detailed in (A). (C). Effect of fenofibrate on the entry of luciferase expressing lentiviruses pseudotyped with SARS-CoV-2 Spike in ACE2-expressing HEK-293T cells. Values are normalized to luciferase expression by mock-treated cells set at 100%. Mean and s.e.m. from two experiments with two replicates. Statistical deviations from 100% were assessed with a one sample t test. (D). Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of MDL 28170, as detailed in (A). (E). Comparison of MDL 28170 activity with entry inhibitors blocking viral endocytosis, such as chloroquine and E-64d, and inhibitors blocking serine protease TMPRSS2, such as camostat. ACE2 expressing HEK-293T cells transfected or not with TMPRSS2 were exposed to SARS-CoV- 2 Spike lentiviruses in the presence of these compounds. Values are normalized to luciferase expression by mock-treated cells set at 100%. Mean and s.e.m. from at least two experiments with two replicates. Statistical deviations from 100% were assessed with a one sample t test. FIGURE 4 | Decreased release of SARS-CoV-2 in the presence of inhibitors with antiviral activity. (A). Viral release to the supernatant in the presence of the indicated compounds added at increasing concentrations 3 days post-infection of Vero E6 cells. SARS-CoV-2 nucleoprotein was detected with an ELISA at concentrations were drugs were nontoxic. Mean and s.e.m. from two experiments. (B). Viral release to the supernatant in the presence of the indicated interferons as described in A. Mean and s.e.m. from one experiment. Frontiers in Pharmacology | www.frontiersin.org 9 March 2021 | Volume 12 | Article 646676 Rodon et al. Plitidepsin potently inhibits SARS-CoV-2 DISCUSSION could happen in vivo upon SARS-CoV-2 infection. The best antiviral compounds found in the present study need to be We have assessed the anti-SARS-CoV-2 activity of clinically tested in adequate animal models. This strategy already helped approved compounds that may exert antiviral effect alone or to confirm the activity of remdesivir against SARS-CoV-2 in combination. Although we were not able to detect any (Williamson et al., 2020), while also questioning the use of remarkable synergy in vitro, further work in more hydroxychloroquine in monotherapy (Maisonnasse et al., 2020). physiological cellular models is needed to address if any Thus, assessing antiviral activity and safety in animal models is key synergism and additivity can be found between identified to identify and advance those compounds with the highest compounds. Combined therapies are key to tackle viral potential to succeed in upcoming clinical trials. In turn, in vitro infections and to reduce the appearance of viral resistance, results confirmed in animal models will provide a rational basis to and that is why identifying synergism and antagonism for perform future clinical trials not only for treatment of SARS-CoV- SARS-CoV-2 antivirals is of key importance (Bobrowski et al., 2-infected individuals, but also for pre-exposure prophylaxis 2020). We have tested more than seventy compounds and their strategies that could avoid novel infections. Prophylaxis could combinations, and verified a potent antiviral effect of be envisioned at a population level or to protect the most hydroxychloroquine and remdesivir, along with plitidepsin, vulnerable groups, and should be implemented until an cathepsin and calpain inhibitors MDL 28170 and NPO, effective vaccine is developed. In particular, orally available nelfinavir mesylate hydrate, interferon-2α, interferon-γ and compounds with proven safety profiles, such as fenofibrate, fenofibrate. These are therefore the most promising agents could represent promising agents. found herein that were able to protect cells from viral-induced cytopathic effect by preventing viral replication. Our findings highlight the utility of using hydroxychloroquine DATA AVAILABILITY STATEMENT and MDL 28170 or other cathepsin inhibitors to block viral entry via the endosomal pathway in kidney cell lines such as Vero E6 or The datasets presented in this study can be found in online HEK-293T. However, the endosomal viral entry route is absent in repositories. The names of the repository/repositories and pulmonary cells (Maisonnasse et al., 2020) and, therefore, accession number(s) can be found below:
http://gisaid. camostat should be considered as the primary inhibitor to org, EPI_ISL_510689. limit SARS-CoV-2 entry in pulmonary tissues or in cells expressing TMPRSS2 (Hoffmann et al., 2020). These findings can explain why randomized clinical trials using ETHICS STATEMENT hydroxychloroquine have failed to show a significant protective effect (Boulware et al., 2020; Cavalcanti et al., 2020). The institutional review board on biomedical research from Nonetheless, in combined therapies, it should be noted that Hospital Germans Trias i Pujol (HUGTiP) approved this agents targeting the alternative endosomal SARS-CoV-2 entry study. The individual who provided the sample to isolate virus route such as hydroxychloroquine or MDL 28170 could be key to gave a written informed consent to participate. stop viral dissemination in other extrapulmonary tissues where viral replication has been already detected (Hanley et al., 2020), and viral entry could take place through this endosomal pathway. AUTHOR CONTRIBUTIONS This could partially explain why in a retrospective observational study including more than 2500 patients, hydroxychloroquine Conceived and designed the experiments: JR, JM-B, DP-Z, JS, BC, treatment showed a significant reduction of in-hospital mortality JV-A, and NI-U. Performed in silico drug modeling: VG and CP. (Arshad et al., 2020). Thus, since alternative routes govern SARS- Performed experiments: JR, JM-B, DP-Z, MN-J, IE, VG, JV-A, CoV-2 viral entry depending on the cellular target (Ou et al., and NI-U. Contributed with critical reagents: CQ and IB. 2020a), effective treatments might be needed to block both Analyzed and interpreted the data: JR, JM-B, DP-Z, MN-J, RP, plasma membrane fusion and endosomal entry to broadly LM, CQ, CP, IE, IB, AV, VG, JC, JB, JS, BC, JV-A, and NI-U. achieve viral suppression. Wrote the paper: JR, JV-A, NI-U. SARS-CoV-2 replication could be effectively blocked using nelfinavir mesylate hydrate, remdesivir and plitidepsin. While nelfinavir showed lower potency, remdesivir and plitidepsin were FUNDING the most potent agents identified. However, remdesivir and plitidepsin are not yet suitable for oral delivery and require The research of CBIG consortium (constituted by IRTA-CReSA, intravenous injection, complicating their clinical use for BSC, and IrsiCaixa) is supported by Grifols pharmaceutical. The prophylaxis. Finally, we also confirmed the antiviral effect of authors also acknowledge the crowdfunding initiative type I and II interferons as well as fenofibrate, which have been #Yomecorono (https://www.yomecorono.com). JS, JV-A and extensively used in the clinic for many years and may therefore NI-U have non-restrictive funding from Pharma Mar to study prove valuable for therapeutic use. the antiviral effect of plitidepsin. The funders had no role in study The data presented herein should be interpreted with caution, design, data collection and analysis, decision to publish, or as the IC50 values of drugs obtained in vitro may not reflect what preparation of the manuscript. Frontiers in Pharmacology | www.frontiersin.org 10 March 2021 | Volume 12 | Article 646676 Rodon et al. Plitidepsin potently inhibits SARS-CoV-2 ACKNOWLEDGMENTS and Drs. Cabrera and Ballana form IrsiCaixa; Pascual-Figal, Lax and Asensio-Lopez MC from “Instituto de Investigación We are grateful to patients at the Hospital Germans Trias i Pujol Biosanitaria IMIB-Arrixaca” of Murcia; and Fernández-Real that donated their samples for research. For his excellent and Barretina from the “Institut d’Investigació Biomèdica de assistance and advice, we thank Jordi Puig from Fundació Girona Dr Josep Trueta” for providing some of the reagents tested. Lluita contra la SIDA. We are most grateful to Lidia Ruiz and the Clinical Sample Management Team of IrsiCaixa for their outstanding sample processing and management, and to M. Pilar SUPPLEMENTARY MATERIAL Armengol and the translational genomics platform team at the Institut de Recerca Germans Trias i Pujol. We truly thank B. The Supplementary Material for this article can be found online at: Trinité for generating the Spike expression plasmid construct used https://www.frontiersin.org/articles/10.3389/fphar.2021.646676/ in this study. We thank Pharma Mar, Rubió Laboratories, Janssen full#supplementary-material. Gautret, P., Lagier, J.-C., Parola, P., Hoang, V. T., Meddeb, L., Mailhe, M., et al. REFERENCES (2020). Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial. Int. J. Antimicrob. Agents Arshad, S., Kilgore, P., Chaudhry, Z. S., Jacobsen, G., Wang, D. D., Huitsing, K., 56, 105949. doi:10.1016/j.ijantimicag.2020.105949 et al. (2020). 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Frontiers in Pharmacology | www.frontiersin.org 12 March 2021 | Volume 12 | Article 646676