Identification of Plitidepsin As Potent Inhibitor of SARS-Cov-2-Induced

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bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055756; this version posted January 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 TITLE: Identification of Plitidepsin as Potent Inhibitor of SARS-CoV-2-Induced 2 Cytopathic Effect after a Drug Repurposing Screen 3 4 5 AUTHORS: Jordi Rodon1, Jordana Muñoz-Basagoiti2,*, Daniel Perez-Zsolt2,*, Marc 6 Noguera-Julian2,7, Roger Paredes2,6, Lourdes Mateu6, Carles Quiñones6, Itziar Erkizia2, 7 Ignacio Blanco5, Alfonso Valencia3,4, Víctor Guallar3,4, Jorge Carrillo2, Julià Blanco2,5,7, 8 Joaquim Segalés8,9, Bonaventura Clotet2,5,7, Júlia Vergara-Alert1,+, Nuria Izquierdo- 9 Useros2,5,+ 10 11 *Equal contribution 12 + Dual senior authorship 13 Corresponding authors: JV-A: [email protected] and NI-U 14 [email protected] 15 16 Affiliations: 1IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), 17 Campus de la UAB, 08193 Bellaterra (Cerdanyola del Vallès), Spain 18 2IrsiCaixa AIDS Research Institute, 08916, Badalona, Spain

19 3Barcelona Supercomputing Center

20 4Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain

21 5Germans Trias i Pujol Research Institute (IGTP), Can Ruti Campus, 08916, Badalona,

22 Spain

23 6Germans Trias i Pujol Hospital, Badalona, Catalonia, Spain

24 7University of Vic–Central University of Catalonia (UVic-UCC), Vic, Spain 25 8UAB, CReSA (IRTA-UAB), Campus de la UAB, 08193 Bellaterra (Cerdanyola del 26 Vallès), Spain 27 9Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, 08193 28 Bellaterra (Cerdanyola del Vallès), Spain 29 30

31 ABSTRACT (234) 32 33 There is an urgent need to identify therapeutics for the treatment of Coronavirus 34 diseases 2019 (COVID-19). Although different antivirals are given for the clinical 35 management of SARS-CoV-2 infection, their efficacy is still under evaluation. Here, we 36 have screened existing drugs approved for human use in a variety of diseases, to 37 compare how they counteract SARS-CoV-2-induced cytopathic effect and viral 38 replication in vitro. Among the potential 72 antivirals tested herein that were previously 2 39 proposed to inhibit SARS-CoV-2 infection, only 18% had an IC50 below 25 µM or 10 40 IU/mL. These included plitidepsin, novel cathepsin inhibitors, nelfinavir mesylate 41 hydrate, interferon 2-alpha, interferon-gamma, fenofibrate, camostat along the well- 42 known remdesivir and chloroquine derivatives. Plitidepsin was the only clinically 43 approved drug displaying nanomolar efficacy. Four of these families, including novel 44 cathepsin inhibitors, blocked viral entry in a cell-type specific manner. Since the most 45 effective antivirals usually combine therapies that tackle the virus at different steps of 46 infection, we also assessed several drug combinations. Although no particular synergy 47 was found, inhibitory combinations did not reduce their antiviral activity. Thus, these 48 combinations could decrease the potential emergence of resistant viruses. Antivirals 49 prioritized herein identify novel compounds and their mode of action, while 50 independently replicating the activity of a reduced proportion of drugs which are mostly 51 approved for clinical use. Combinations of these drugs should be tested in animal 52 models to inform the design of fast track clinical trials. 53 54 55 Keywords: SARS-CoV-2, antivirals, plitidepsin, synergy

56 INTRODUCTION 57 A novel betacoronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS- 58 CoV-2), is causing a respiratory disease pandemic that began in Wuhan, China, in 59 November 2019, and has now spread across the world 1. To date, remdesivir is the only 60 approved antiviral drug for the specific treatment of this coronavirus infectious disease 61 2019 or COVID-19 2,3. However, several drugs are being used in the frontline of clinical 62 management of SARS-CoV-2-infected individuals in hospitals all around the world, to 63 try to avoid the development of the COVID-19 associated pneumonia, which can be 64 fatal. By the end of December 2020, almost 1.7 million people had died from COVID- 65 19, and over 75 million cases have been reported (https://covid19.who.int). 66 67 Although different drug regimens are being applied to hospitalized patients, no clinical 68 study has evidenced their efficacy yet. Under this scenario, initiatives launched by the 69 World Health Organization (WHO), such as the SOLIDARITY study that has compared 70 remdesivir, hydroxychloroquine, ritonavir/lopinavir and ritonavir/lopinavir plus ß- 71 interferon regimes, have been of critical importance to prioritize the use of the most 72 active compounds 4. Unfortunately, although remdesivir has proven efficacy in 73 randomized controlled trials 2,3, a recent update of the WHO clinical trial has failed to 74 detect any effect on overall mortality, initiation of ventilation and duration of hospital 75 stay with any of the antivirals tested 5. Thus, there is an urgent need to identify novel 76 therapeutic approaches for individuals with COVID-19 developing severe disease and 77 fatal outcomes. 78 79 In this report we present a prioritized list of effective compounds with proven antiviral 80 efficacy in vitro to halt SARS-CoV-2 replication. Compounds were analyzed depending 81 on their expected mechanism of action, to identify candidates tackling diverse steps of 82 the viral life cycle. SARS-CoV-2 entry requires viral binding and spike protein 83 activation via interaction with the cellular receptor ACE2 and the cellular protease 84 TMPRSS26, a mechanism favored by viral internalization via endocytosis. Interference 85 with either of these processes has proven to decrease SARS-CoV-2 infectivity6,7, and 86 therefore, inhibitors targeting viral entry may prove valuable. In addition, SARS-CoV-2 87 enters into the cells via endocytosis and accumulates in endosomes where cellular 88 cathepsins can also prime the spike protein and favor viral fusion upon cleavage 6,8,9, 89 providing additional targets for antiviral activity. Once SARS-CoV-2 fuses with cellular

90 membranes, it triggers viral RNA release into the cytoplasm, where polyproteins are 91 translated and cleaved by proteases 10. This leads to the formation of an RNA replicase- 92 transcriptase complex driving the production of several negative-stranded RNA via both 93 replication and transcription 10. Numerous negative-stranded RNAs transcribe into 94 messenger RNA genomes, allowing for the translation of viral nucleoproteins, which 95 assemble in viral capsids at the cytoplasm 10. These capsids then bud into the lumen of 96 endoplasmic reticulum (ER)-Golgi compartments, where viruses are finally released 97 into the extracellular space by exocytosis. Potentially, any of these viral cycle steps 98 could be targeted with antivirals, so we have thus searched for these compounds as well. 99 100 Finally, as the most effective antiviral treatments are usually based on combined 101 therapies that tackle distinct steps of the viral life cycle, we also tested the active 102 compounds in combination. These combinations may be critical to abrogate the 103 potential emergence of resistant viruses and to increase antiviral activity, enhancing the 104 chances to improve clinical outcome. 105

106 MATERIAL & METHODS 107 Ethics statement. The institutional review board on biomedical research from Hospital 108 Germans Trias i Pujol (HUGTiP) approved this study. The individual who provided the 109 sample to isolate virus gave a written informed consent to participate. 110 111 Cell Cultures. Vero E6 cells (ATCC CRL-1586) were cultured in Dulbecco’s modified 112 Eagle medium, (DMEM; Lonza) supplemented with 5% fetal calf serum (FCS; 113 EuroClone), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM glutamine (all 114 ThermoFisher Scientific). HEK-293T (ATCC repository) were maintained in DMEM 115 with 10% fetal bovine serum, 100 IU/mL penicillin and 100 μg/mL streptomycin (all 116 from Invitrogen). HEK-293T overexpressing the human ACE2 were kindly provided by 117 Integral Molecular Company and maintained in DMEM (Invitrogen) with 10% fetal 118 bovine serum, 100 IU/mL penicillin and 100 μg/mL streptomycin, and 1 μg/mL of 119 puromycin (all from Invitrogen). TMPRSS2 human plasmid (Origene) was transfected 120 using X-tremeGENE HP Transfection Reagent (Merck) on HEK-293T overexpressing 121 the human ACE2 and maintained in the previously described media containing 1 mg/ml 122 of geneticin (Invitrogen) to obtain TMPRSS2/ACE2 HEK-293T cells. 123 124 Virus isolation, titration and sequencing. SARS-CoV-2 was isolated from a 125 nasopharyngeal swab collected from an 89-year-old male patient giving informed 126 consent and treated with Betaferon and hydroxychloroquine for 2 days before sample 127 collection. The swab was collected in 3 mL medium (Deltaswab VICUM) to reduce 128 viscosity and stored at -80ºC until use. Vero E6 cells were cultured on a cell culture 129 flask (25 cm2) at 1.5 x 106 cells overnight prior to inoculation with 1 mL of the

130 processed sample, for 1 h at 37ºC and 5% CO2. Afterwards, 4 mL of 2% FCS- 131 supplemented DMEM were supplied and cells were incubated for 48 h. Supernatant was 132 harvested, centrifuged at 200 x g for 10 min to remove cell debris and stored at -80ºC. 133 Cells were assessed daily for cytopathic effect and the supernatant was subjected to 134 viral RNA extraction and specific RT-qPCR using the SARS-CoV-2 UpE, RdRp and N 135 assays 11. The virus was propagated for two passages and a virus stock was prepared 136 collecting the supernatant from Vero E6. 137 138 Viral RNA was extracted directly from the virus stock using the Indimag Pathogen kit 139 (Indical Biosciences) and transcribed to cDNA using the PrimeScript™ RT reagent Kit

140 (Takara) using oligo-dT and random hexamers, according to the manufacturer's 141 protocol. DNA library preparation was performed using SWIFT amplicon SARS-CoV-2 142 panel (Swift Biosciences). Sequencing ready libraries where then loaded onto Illumina 143 MiSeq platform and a 300bp paired-end sequencing kit. Sequence reads were quality 144 filtered and adapter primer sequences were trimmed using trimmomatic. Amplification 145 primer sequences were removed using cutadapt 12. Sequencing reads were then mapped 146 against coronavirus reference (NC_045512.2) using bowtie2 tool 13. Consensus 147 genomic sequence was called from the resulting alignment at a 18x1800x879 average 148 coverage using samtools 14. Genomic sequence was deposited at GISAID repository 149 (http://gisaid.org) with accession ID EPI_ISL_510689. 150 151 Pseudovirus production. HIV-1 reporter pseudoviruses expressing SARS-CoV-2 Spike 152 protein and luciferase were generated using two plasmids. pNL4-3.Luc.R-.E- was 153 obtained from the NIH AIDS repository. SARS-CoV-2.SctΔ19 was generated (Geneart) 154 from the full protein sequence of SARS-CoV-2 spike with a deletion of the last 19 155 amino acids in C-terminal, human-codon optimized and inserted into pcDNA3.4-TOPO 156 15. Spike plasmid was transfected with X-tremeGENE HP Transfection Reagent 157 (Merck) into HEK-293T cells, and 24 hours post-transfection, cells were transfected 158 with pNL4-3.Luc.R-.E-. Supernatants were harvested 48 hours later, filtered with 0.45 159 µM (Millex Millipore) and stored at -80ºC until use. Control pseudoviruses were 160 obtained by replacing the spike plasmid by a VSV-G plasmid (kindly provided by Dr. 161 Andrea Cimarelli). The p24gag content of all viruses was quantified using an ELISA 162 (Perkin Elmer) and viruses were titrated in HEK-293T overexpressing the human 163 ACE2. 164 165 Antivirals & compounds. The complete list of compounds used for this study and 166 vendors are shown in Supplementary Tables 1 to 5. Drugs were used at concentrations 167 ranging from 100 µM to 0.0512 nM at 5-fold serial dilutions. NPOs were used at 168 concentrations ranging from 10 µM to 0.00512 nM at 5-fold serial dilutions. Plitidepsin 169 was also assayed at concentrations ranging from 10 µM to 0.5 nM at 3-fold dilutions. 170 Interferons were assayed at concentrations ranging from 104 to 0.0005 IU/ml at 5-fold 171 serial dilutions. When two drugs were combined, each one was added at a 1:1 molar 172 ratio at concentrations ranging from 100 µM to 0.0512 nM at 5-fold serial dilutions. In

173 combination with other drugs, plitidepsin was also assayed at concentrations ranging 174 from 10 µM to 0.5 nM at 3-fold dilutions. 175 176 Antiviral activity. Increasing concentrations of antiviral compounds were added to Vero 177 E6 cells and immediately after, we added 101.8 TCID50/mL of SARS-CoV-2, a 178 concentration that achieves a 50% of cytopathic effect. Untreated non-infected cells and 179 untreated virus-infected cells were used as negative and positive controls of infection, 180 respectively. To detect any drug-associated cytotoxic effect, Vero E6 cells were equally 181 cultured in the presence of increasing drug concentrations, but in the absence of virus. 182 Cytopathic or cytotoxic effects of the virus or drugs were measured 3 days after 183 infection, using the CellTiter-Glo luminescent cell viability assay (Promega). 184 Luminescence was measured in a Fluoroskan Ascent FL luminometer (ThermoFisher 185 Scientific). 186 187 IC50 calculation and statistical analysis. Response curves of compounds or their mixes 188 were adjusted to a non-linear fit regression model, calculated with a four-parameter 189 logistic curve with variable slope. Cells not exposed to the virus were used as negative 190 controls of infection, and were set as 100% of viability to normalize data and calculate 191 the percentage of cytopathic effect. Statistical differences from 100% were assessed 192 with a one sample t test. All analyses and figures were generated with the GraphPad 193 Prism v8.0b Software. 194 195 In silico drug modeling. We performed Glide docking using an in-house library of all 196 approved drug molecules on the 3CL protease of SARS-CoV-2. For this, two different 197 receptors were used, the 6LU7 pdb structure, after removing the covalently bound 198 inhibitor, and a combination of two crystals from the Diamond collection 199 (https://www.diamond.ac.uk/covid-19). Receptors were prepared with the Schrodinger's 200 protein wizard and Glide SP docking was performed with two different hydrogen bond 201 constraints: Glu16 and His163 (with epsilon protonation); we enforced single 202 constraints and also attempted the combination of both. The best 11 molecules, based on 203 Glides’s docking score were selected. Top docking scores, however, did not exceed -9, 204 indicating poor potential binding. 205

206 Pseudovirus assay. HEK-293T overexpressing the human ACE2 and TMPRSS2 were 207 used to test antivirals at the concentrations found to be effective for SARS-CoV-2 208 without toxicity, which were the following: 5 µM for niclosamide; 10 µM for 209 chloroquine, chlorpromazine, ciclesonide, MDL 28170 and fenofibrate; 20 µM for 210 hydroxychloroquine, CA-074-Me and arbidol HCl; 25 µM for E-64d; 50 µM for 211 Baricitinib; 100 µM for Amantadine, NB-DNJ, 3’ sialyl-lactose Na salt, Tofacitinib, 212 and Camostat mesylate; 1000 µM for methyl-β-cyclodextrin, and 12,5 mg/ml for AAT. 213 A constant pseudoviral titer was used to pulse cells in the presence of the drugs. At 48h 214 post-inoculation, cells were lysed with the Glo Luciferase system (Promega). 215 Luminescence was measured with an EnSight Multimode Plate Reader (Perkin Elmer). 216 217 SARS-CoV-2 detection in the supernatant of infected cells. Viral accumulation in the 218 supernatant of Vero E6 cells infected as described previously in the presence of 219 increasing concentrations of the indicated antiviral compounds was measured at day 3 220 post-infection. The amount of SARS-CoV-2 nucleoprotein released to the supernatant 221 was measured with an ELISA (SinoBiologicals), according to the manufacturer’s 222 protocol. 223

224 RESULTS 225 We have tested the antiviral activity of different clinically available compounds and 226 their combinations by assessing their ability to inhibit viral induced cytopathic effect in 227 vitro. Our strategy was circumscribed mostly to compounds approved for clinical use, 228 since they are ideal candidates for entering into fast track clinical trials. Drug selection 229 criteria first focused on compounds already being tested in clinical trials, along with 230 well-known human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) 231 inhibitors, as well as other compounds suggested to have potential activity against 232 SARS-CoV-2 in molecular docking analysis or in vitro assays. 233 234 We first assessed the activity of 16 compounds with hypothetical capacity to inhibit 235 viral entry, and then we focused on 22 drugs thought to block viral replication upon 236 SARS-CoV-2 fusion. Molecular docking studies provided an additional 11 candidates, 237 which were predicted to inhibit the SARS-CoV-2 main protease. Finally, 23 compounds 238 with unknown mechanism of action were also assessed. By these means, we have 239 compared 72 drugs and 28 of their combinations for their capacity to counteract SARS- 240 CoV-2-induced cytopathic effect in vitro. 241 242 1. Antiviral activity of compounds that potentially inhibit viral entry 243 We first tested compounds that could have an effect before viral entry by impairing 244 virus-cell fusion (Supp. Table 1). We confirmed the inhibitory effect of 245 hydroxychloroquine against SARS-CoV-2-induced cellular cytotoxicity on Vero E6 246 cells 16. As shown in Fig. 1A, this drug was able to inhibit viral-induced cytopathic 247 effects at concentrations where no cytotoxic effects of the drug were observed, as 248 previously reported16,17. Since hydroxychloroquine was first administered in 249 combination with the antibiotic azithromycin 22, which induces antiviral responses in 250 bronchial epithelial cells 23, we tested this antibiotic alone and in combination (Fig. 1A), 251 but found a similar activity to that of the chloroquine derivative alone (Fig. 1A). Indeed, 252 this was also the case when we tested hydroxychloroquine in combination with different 253 HIV-1 protease inhibitors and other relevant compounds currently being tested in 254 clinical trials (Supp. Table 2). 255 256 Additional Food and Drug Administration (FDA)-approved compounds previously used 257 to abrogate viral entry via clathrin-mediated endocytosis such as amantadine or

258 chlorpromazine were also tested in this SARS-CoV-2-induced cytotoxicity assay 259 (Supp. Table 1). Yet, we did not find any prominent effect of these agents; only a 260 partial inhibition at 100 μM for amantadine (Fig. 1B). The broad cathepsin B/L 261 inhibitor E64-d also showed partial inhibitory activity (Fig. 1B). E64-d exerts activity 262 against viruses cleaved by cellular cathepsins upon endosomal internalization, as 263 previously described using pseudotyped SARS-CoV-26. While these results could not 264 be confirmed using the specific cathepsin B inhibitor CA-074-Me due to drug- 265 associated toxicity (Supp. Table 1), none of these cathepsin inhibitors is approved for 266 clinical use. It has also been suggested that hydroxychloroquine could block SARS- 267 CoV-2 spike interaction with GM1 gangliosides 28. GM1 gangliosides are enriched in 268 cholesterol domains of the plasma membrane and have been previously shown to bind 269 to SARS-CoV spike protein 29. This mode of viral interaction is aligned with the 270 capacity of methyl-beta cyclodextrin, which depletes cholesterol from the plasma 271 membrane to abrogate SARS-CoV-2 induced cytopathic effect (Fig. 1B), as previously 272 reported for SARS-CoV 29. Removal of cholesterol redirected ACE2 receptor to other 273 domains, but did not alter the expression of the viral receptor 29. Moreover, NB-DNJ, an 274 inhibitor of ganglioside biosynthesis pathway, also decreased SARS-CoV-2 cytopathic 275 effect (Fig. 1B). These results highlight the possible role of gangliosides in viral 276 binding, although in soluble competition the polar head group of GM3 ganglioside (3’ 277 Sialyllactose) was not able to reduce viral-induced cytopathic effect (Supp. Table 1). 278 Of note, previously reported antiviral agents such as the autophagy BECN1-stabilizing 279 compounds niclosamide or ciclesonide30,31, the membrane lipid intercalating compound 280 arbidol32 and the two JAK inhibitors baricitinib and tofacitinib34,35 failed to protected 281 Vero E6 cells from SARS-CoV-2 induced cytopathic effect (Supp. Table 1). We next 282 tested camostat, a serine protease inhibitor with capacity to abrogate SARS-CoV-2 283 Spike priming on the plasma membrane of human pulmonary cells and avoid viral 284 fusion 6. Camostat showed no antiviral effect on Vero E6 cells (Fig. 1C), what indicates 285 that the alternative viral endocytic route is the most prominent entry route in this renal 286 cell type. A broader cellular protease inhibitor such as alpha-1 antitrypsin (AAT), used 287 to treat severe AAT human deficiency and currently under clinical study for COVID-19 288 due to its anti-inflammatory potential, has already shown capacity to limit SARS-CoV-2 289 entry in cells expressing TMPRSS2 36,37. When we tested AAT antiviral efficacy on 290 Vero E6, AAT exerted an effect but required high concentrations that will most likely 291 rely on the activity of these proteases in the endosomal route (Fig. 1C). 10

292 293 To confirm that identified compounds listed in Supp. Table 1 specifically inhibit the 294 viral entry step, we employed a luciferase-based assay using pseudotyped lentivirus 295 expressing the spike protein of SARS-CoV-2, which allows to detect viral fusion on 296 HEK-293T cells transfected with ACE2. As a control, we used lentiviruses pseudotyped 297 with a VSV glycoprotein, where no entry inhibition above 20% was detected for any of 298 the drugs tested (data not shown). In sharp contrast, SARS-CoV-2 pseudoviruses were 299 effectively blocked by most of the drugs previously tested on Vero E6 with wild-type 300 virus (Fig. 1D). The main differences were observed with CA-074-Me, ciclesonide and 301 arbidol. These compounds showed a partial blocking effect on ACE2 HEK-293T cells 302 that was not obvious when using replication competent SARS-CoV-2 on Vero E6 303 (Supp. Fig. 1). In addition, NB-DNJ failed to block viral entry (Fig. 1D). Overall, using 304 alternative SARS-CoV-2 viral systems, we could identify chloroquine derivatives, 305 cathepsin inhibitors and cholesterol depleting agents as the most promising candidates 306 to block SARS-CoV-2 endocytosis in Vero E6 and HEK-293T cells transfected with

307 ACE2. However, chloroquine derivatives were the only ones that displayed an IC50 308 below 25 µM (Table 1), and were also active abrogating pseudoviral entry into HEK- 309 293T cells expressing ACE2 (Fig. 1E). Although camostat failed to inhibit viral fusion 310 on ACE2 HEK-293T cells (Fig. 1E), its activity was rescued when these cells were 311 transfected with TMPRSS2. The opposite effect was observed for chloroquine, which 312 reduced its inhibitory activity on TMPRSS2 transfected cells (Fig. 1E). Our results 313 highlight that alternative routes govern SARS-CoV-2 viral entry and these pathways 314 vary depending on the cellular target. Thus, effective treatments may need to block both 315 plasma membrane fusion and endosomal routes to fully achieve viral suppression. 316 317 2. Antiviral activity of compounds that potentially inhibit post-entry steps. 318 In our search for antivirals inhibiting post-viral entry steps, we first focused on 319 remdesivir, which has in vitro activity against SARS-CoV-2 after viral entry 17 and has 320 already been approved for the treatment of COVID-19 by the FDA and EMA. We 321 further confirmed the in vitro capacity of remdesivir to inhibit SARS-CoV-2-induced

322 cytopathic effect on Vero E6 (Fig. 2A). The IC50 value of this drug in repeated 323 experiments was always below 10 µM (Supp. Table 2). In combination with increasing 324 concentrations of hydroxychloroquine, however, remdesivir did not significantly 325 modified its own antiviral effect (Fig. 2B). This was also the case for other antivirals

326 tested in combination (Supp. Table 2). Of note, other RNA polymerase inhibitors such 327 as galdesivir, which was proposed to tightly bind to SARS-CoV-2 RNA-dependent 328 RNA polymerase 38, showed no antiviral effect (Supp. Table 3). Favipiravir, approved 329 by the National Medical Products Administration of China as the first anti-COVID-19 330 drug in China 39, showed only partial inhibitory activity at the non-toxic concentration 331 of 100 µM (Supp. Table 3). 332 333 We also assessed clinically approved protease inhibitors with potent activity against 334 HIV-1. However, none of the HIV-1 protease inhibitors detailed in Supp. Table 3 335 showed remarkable protective antiviral activity against SARS-CoV-2 infection on Vero

336 E6 cells, with the exception of nelfinavir mesylate hydrate, which showed an IC50 value 337 below 10 µM (Supp. Table 3 and Fig. 2C). Lopinavir and tipranavir inhibited SARS- 338 CoV-2-induced cytopathic effect at the non-toxic concentration of 20 μM, and 339 amprenavir exhibited activity at the non-toxic concentration of 100 µM (Fig. 2C). 340 Darunavir, which has been tested in clinical trials, showed partial inhibitory activity at 341 100 μM, although this concentration had 8.5 ± 6.2 % of cytotoxicity associated (Fig. 342 2C). Of note, we tested HIV-1 reverse transcriptase inhibitors such as tenofovir 343 disoproxil fumarate, emtricitabin, tenofovir alafenamide, and their combinations, but 344 they also failed to show any antiviral effect against SARS-CoV-2 (Supp. Fig. 1). These 345 results indicate that future clinical trials should contemplate the limited antiviral effect 346 displayed by these anti-HIV-1 inhibitors against SARS-CoV-2 in vitro. 347 348 We also assessed the inhibitory capacity of HCV inhibitors, but none showed any 349 antiviral activity (Supp. Table 3). Of note, exogenous interferons 2 alpha and gamma 350 displayed antiviral activity against SARS-CoV-2 (Supp. Table 3). In light of these 351 results, we tested the inhibitory effect of the TLR 7 agonist vesatolimod that triggers 352 interferon production. Although this agonist was not able to protect from the viral- 353 induced cytopathic effect on Vero E6 (Supp. Table 3), as expected since it is an 354 interferon-producer deficient cell line 40, it could still be useful in other competent 355 cellular targets. Since severe COVID-19 patients display impaired interferon responses 356 41, these strategies may be valuable to avoid disease complication. In addition, we also 357 assessed several compounds with the best computational docking scores among 358 approved drugs against the 3CL protease of SARS-CoV-2, but none of them were 359 effective to protect Vero E6 from viral induced cytopathic effect (Supp. Table 4). 12

360 361 The most potent antiviral tested was plitidepsin (Fig. 2D), which targets the eukaryotic 362 Elongation Factor 1A2 (eEF1A2) and has been previously used for the treatment of

363 multiple myeloma. The mean IC50 value of this drug in repeated experiments was 364 always in nM concentrations (Supp. Table 3). In combination with other active

365 antivirals, we did not observe a reduction on IC50 values (Supp. Table 2). This result 366 indicates no significant synergy, but also highlights the possibility of using plitidepsin 367 without reducing its antiviral activity in combined therapies (Fig. 2D), what could be 368 relevant to avoid possible selection of resistant viruses. Overall, plitidepsin showed the

369 lowest IC50 values of all the compounds tested in this in vitro screening (Table 1). 370 371 3. Antiviral activity of compounds with unknown mechanism of action. 372 We also assessed the inhibitory capacity of several inhibitors and broad anti-bacterial, 373 anti-parasitic, anti-malarial, anti-influenza and anti-fungal compounds, along with other 374 pharmacological agents previously suggested to interfere with SARS-CoV-2 infection 375 (Supp. Table 5). Such was the case of ivermectin, an FDA-approved broad spectrum 376 anti-parasitic agent previously reported to inhibit the replication of SARS-CoV-2 in 377 vitro as measured by RNA accumulation 42. 378 379 However, among these potential antivirals, only three types of molecules exerted 380 detectable antiviral activity in our assay: itraconazole, fenofibrate, and calpain and 381 cathepsin inhibitors such as MDL 28170 and NPO compounds. Itraconazole, an 382 antifungal that may interfere with internal SARS-CoV-2 budding within infected cells 43 383 , displayed an IC50 value of 80 µM (Fig. 3A and Supp. Table 5). Fenofibrate is 384 clinically used to treat dyslipidemia via activation of PPARα, and also inhibited the 385 cytopathic effect exerted by SARS-CoV-2 on Vero E6 at 20 µM (Fig. 3B and Supp. 386 Table 5). As fenofibrate is a regulator of cellular lipid metabolism, we made use of the 387 luciferase-based viral entry assay to try to elucidate its mode of action. When 388 lentiviruses pseudotyped with the spike protein of SARS-CoV-2 were added to ACE2- 389 expressing HEK-293T cells in the presence of fenofibrate, viral entry was abrogated 390 (Fig. 3C). The most potent agent found was MDL 28170, a calpain III inhibitor in a pre- 391 clinical stage of development that displayed activity in the nanomolar range (Fig. 3D 392 and Supp. Table 5), as previously identified in vitro 44. Moreover, three out of four 393 different calpain and cathepsin inhibitors named NPO showed potent antiviral activity 13

394 too (Supp. Figure 2). Of note, in combination with other active antivirals, we did not

395 observe a reduction on IC50 values of MDL 28170 (Supp. Table 2). 396 397 Inhibitors of calpains, which are cysteine proteases, might impair the activity of viral 398 proteases like 3CL (main protease) and PLpro (papain-like protease) 44,45. However, 399 calpain inhibitors may also inhibit cathepsin B-mediated processing of viral spike 400 proteins or glycoproteins, including SARS-CoV and Ebola 45,46. To understand the 401 mechanisms of action of calpain and cathepsin inhibitors such as MDL 28170, we 402 added lentiviruses pseudotyped with the spike protein of SARS-CoV-2 to ACE2- 403 expressing HEK-293T cells and the same cells also expressing TMPRSS2 in the 404 presence of this drug. Importantly, MDL 28170 only blocked viral entry in ACE2- 405 expressing cells (Fig. 3E). This result indicates that MDL 28170 blocks cathepsins that 406 are implicated in SARS-CoV-2 entry via the alternative endosomal pathway, as 407 described for chloroquine derivatives and E-64d (Fig. 3E), which are all active when 408 TMPRSS2 is not present and their inhibitor camostat displays no activity (Fig. 3E). 409 410 In conclusion, among the 72 compounds and their 28 combinations tested herein for 411 their potential capacity to abrogate SARS-CoV-2 cytopathic effect, we only found 13 412 compounds with antiviral activity including camostat, and only eight types of these 2 413 drugs had an IC50 below 25 µM or 10 IU/mL (Table 1). These eight families of 414 compounds were able to abrogate SARS-CoV-2 release to the supernatant in a dose 415 dependent manner (Fig. 4), indicating that the reduction in the cytopathic effect that we 416 had measured in cells correlates with viral production. As these eight families of 417 compounds tackle different steps of the viral life cycle, they could be tested in 418 combined therapies to abrogate the potential emergence of resistant viruses. 419

420 DISCUSSION 421 We have assessed the anti-SARS-CoV-2 activity of clinically approved compounds that 422 may exert antiviral effect alone or in combination. Although we were not able to detect 423 any remarkable synergy in vitro, combined therapies are key to tackle viral infections 424 and to reduce the appearance of viral resistance. We have tested more than seventy 425 compounds and their combinations, and verified a potent antiviral effect of 426 hydroxychloroquine and remdesivir, along with plitidepsin, cathepsin and calpain 427 inhibitors MDL 28170 and NPO, nelfinavir mesylate hydrate, interferon 2α, interferon- 428 γ and fenofibrate. These are therefore the most promising agents found herein that were 429 able to protect cells from viral-induced cytopathic effect by preventing viral replication. 430 431 Our findings highlight the utility of using hydroxychloroquine and MDL 28170 or other 432 cathepsin inhibitors to block viral entry via the endosomal pathway in kidney cell lines 433 such as Vero E6 or HEK-293T. However, the endosomal viral entry route is absent in 434 pulmonary cells18 and, therefore, camostat should be considered as the primary inhibitor 435 to limit SARS-CoV-2 entry in pulmonary tissues or in cells expressing TMPRSS26. 436 These findings can explain why randomized clinical trials using hydroxychloroquine 437 have failed to show a significant protective effect 20,21. Nonetheless, in combined 438 therapies, it should be noted that agents targeting the alternative endosomal SARS- 439 CoV-2 entry route such as hydroxychloroquine or MDL 28170 could be key to stop 440 viral dissemination in other extrapulmonary tissues where viral replication has been 441 already detected 47, and viral entry could take place through this endosomal pathway. 442 This could partially explain why in a retrospective observational study including more 443 than 2500 patients, hydroxychloroquine treatment showed a significant reduction of in- 444 hospital mortality 48. Thus, since alternative routes govern SARS-CoV-2 viral entry 445 depending on the cellular target 49, effective treatments might be needed to block both 446 plasma membrane fusion and endosomal entry to broadly achieve viral suppression. 447 448 SARS-CoV-2 replication could be effectively blocked using nelfinavir mesylate 449 hydrate, remdesivir and plitidepsin. While nelfinavir showed lower potency, remdesivir 450 and plitidepsin were the most potent agents identified. However, remdesivir and 451 plitidepsin are not yet suitable for oral delivery and require intravenous injection, 452 complicating their clinical use for prophylaxis. Finally, we also confirmed the antiviral

453 effect of type I and II interferons as well as fenofibrate, which have been extensively 454 used in the clinic for many years and may therefore prove valuable for therapeutic use. 455

456 The data presented herein should be interpreted with caution, as the IC50 values of drugs 457 obtained in vitro may not reflect what could happen in vivo upon SARS-CoV-2 458 infection. The best antiviral compounds found in the present study need to be tested in 459 adequate animal models. This strategy already helped to confirm the activity of 460 remdesivir against SARS-CoV-2 50, while also questioning the use of 461 hydroxychloroquine in monotherapy 18. Thus, assessing antiviral activity and safety in 462 animal models is key to identify and advance those compounds with the highest 463 potential to succeed in upcoming clinical trials. In turn, in vitro results confirmed in 464 animal models will provide a rational basis to perform future clinical trials not only for 465 treatment of SARS-CoV-2-infected individuals, but also for pre-exposure prophylaxis 466 strategies that could avoid novel infections. Prophylaxis could be envisioned at a 467 population level or to protect the most vulnerable groups, and should be implemented 468 until an effective vaccine is developed. In particular, orally available compounds with 469 proven safety profiles, such as fenofibrate, could represent promising agents. 470

471 ACKNOWLEDGEMENTS 472 We are grateful to patients at the Hospital Germans Trias i Pujol that donated their 473 samples for research. For his excellent assistance and advice, we thank Jordi Puig from 474 Fundació Lluita contra la SIDA. We are most grateful to Lidia Ruiz and the Clinical 475 Sample Management Team of IrsiCaixa for their outstanding sample processing and 476 management, and to M. Pilar Armengol and the translational genomics platform team at 477 the Institut de Recerca Germans Trias i Pujol. We truly thank B. Trinité for generating 478 the Spike expression plasmid construct used in this study. We thank Pharma Mar, Rubió 479 Laboratories, Janssen and Drs. Cabrera and Ballana form IrsiCaixa; Pascual-Figal, Lax 480 and Asensio-Lopez MC from “Instituto de Investigación Biosanitaria IMIB-Arrixaca” 481 of Murcia; and Fernández-Real and Barretina from the “Institut d'Investigació 482 Biomèdica de Girona Dr. Josep Trueta” for providing some of the reagents tested. 483 FINANCIAL SUPPORT 484 The research of CBIG consortium (constituted by IRTA-CReSA, BSC, & IrsiCaixa) is 485 supported by Grifols pharmaceutical. The authors also acknowledge the crowdfunding 486 initiative #Yomecorono (https://www.yomecorono.com). JS, JVA and NIU have non- 487 restrictive funding from Pharma Mar to study the antiviral effect of plitidepsin. The 488 funders had no role in study design, data collection and analysis, decision to publish, or 489 preparation of the manuscript. 490 COMPETING INTEREST 491 A patent application based on this work has been filed (EP20382821.5). The authors 492 declare that no other competing financial interests exist. 493

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629 TABLES 630

631 Table 1. Compounds with antiviral activity grouped in colors depending on their IC50

632 values on Vero E6. Green color highlights compounds with the lower IC50 in the nM 633 range, while orange color shows compounds in the µM or 102 IU/mL range. 634 635 FIGURES 636 Figure 1. Antiviral activity of entry inhibitors against SARS-CoV-2. A. Antiviral 637 activity of hydroxychloroquine and azithromycin. Cytopathic effect on Vero E6 cells 638 exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing 639 concentrations of hydroxychloroquine, azithromycin, and their combination. Drugs 640 were used at a concentration ranging from 0.0512 nM to 100 µM. When combined, 641 each drug was added at the same concentration. Non-linear fit to a variable response 642 curve from one representative experiment with two replicates is shown (red lines),

643 excluding data from drug concentrations with associated toxicity. The particular IC50 644 value of this graph is indicated. Cytotoxic effect on Vero E6 cells exposed to increasing 645 concentrations of drugs in the absence of virus is also shown (grey lines). B. Cytopathic 646 effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the 647 presence of increasing concentrations of amantadine, a clathrin-mediated endocytosis 648 inhibitor, E-64d, a pan-cathepsin inhibitor acting downstream once viruses are 649 internalized in endosomes, NB-DNJ, an inhibitor of ganglioside biosynthesis and 650 methyl-β-cyclodextrin, a cholesterol-depleting agent. All drugs were used at a 651 concentration ranging from 0.0512 nM to 100 µM aside from methyl-β-cyclodextrin, 652 which was used 10 times more concentrated. Non-linear fit to a variable response curve 653 from one experiment with two replicates is shown (red lines). Cytotoxic effect on Vero 654 E6 cells exposed to increasing concentrations of drugs in the absence of virus is also 655 shown (grey lines). C. Cytopathic effect on Vero E6 cells exposed to a fixed 656 concentration of SARS-CoV-2 in the presence of increasing concentrations of camostat, 657 a TMPRSS2 inhibitor, and ATT, an alpha-1 antyitrypsin, a broad cellular protease 658 inhibitor, as described in A. D. Effect of entry inhibitors on luciferase expression of 659 reporter lentiviruses pseudotyped with SARS-CoV-2 Spike in ACE2 expressing HEK- 660 293T cells. Values are normalized to luciferase expression by mock-treated cells set at 661 100%. Mean and s.e.m. from two experiments with one to three replicates. Cells were 662 exposed to fixed amounts of SARS-CoV-2 Spike lentiviruses in the presence of a non- 21

663 toxic constant concentration of the drugs tested on Vero E6. Significant statistical 664 deviations from 100% were assessed with a one sample t test. E. Comparison of entry 665 inhibitors blocking viral endocytosis, such as chloroquine, with inhibitors blocking 666 serine protease TMPRSS2 expressed on the cellular membrane, such as camostat, on 667 different cell lines. ACE2 expressing HEK-293T cells transfected or not with 668 TMPRSS2 were exposed to SARS-CoV-2 Spike lentiviruses as described in B. Values 669 are normalized to luciferase expression by mock-treated cells set at 100%. Mean and 670 s.e.m. from at least two representative experiments with two replicates. Statistical 671 deviations from 100% were assessed with a one sample t test. 672 673 Figure 2. Antiviral activity of post-entry inhibitors. A. Cytopathic effect on Vero E6 674 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing 675 concentrations of Remdesivir. Drug was used at a concentration ranging from 0.0512 676 nM to 100 µM. Non-linear fit to a variable response curve from one representative 677 experiment with two replicates is shown (red lines), excluding data from drug

678 concentrations with associated toxicity. The particular IC50 value of this graph is 679 indicated. Cytotoxic effect on Vero E6 cells exposed to increasing concentrations of 680 drugs in the absence of virus is also shown (grey lines). B. Cytopathic effect on Vero E6 681 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing 682 concentrations of remdesivir and its combination with hydroxychloroquine, as detailed 683 in A. Drugs in combination were used at a concentration ranging from 0.0512 nM to 684 100 µM (left panel). C. Cytopathic effect on Vero E6 cells exposed to a fixed 685 concentration of SARS-CoV-2 in the presence of increasing concentrations of protease 686 inhibitors against HIV-1. Nelfinavir mesylate hydrate was the only drug with activity. 687 Inhibitors were used at a concentration ranging from 0.0512 nM to 100 µM. The

688 particular IC50 value of this graph is indicated D. Cytopathic effect on Vero E6 cells 689 exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing 690 concentrations of plitidepsin and its combinations with hydroxychloroquine and 691 remdesivir. When combined, each drug was added at the same concentration. Drugs

692 were used at a concentration ranging from 0.5 nM to 10 µM. The particular IC50 value 693 of these graphs is indicated. 694 695 Figure 3. Antiviral activity of inhibitors with unknown mechanism of action. A. 696 Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in

697 the presence of increasing concentrations of Itraconazole. Drug was used at a 698 concentration ranging from 0.0512 nM to 100 µM . Non-linear fit to a variable response 699 curve from one representative experiment with two replicates is shown (red lines),

700 excluding data from drug concentrations with associated toxicity. The particular IC50 701 value of this graph is indicated. Cytotoxic effect on Vero E6 cells exposed to increasing 702 concentrations of drugs in the absence of virus is also shown (grey lines). B. Cytopathic 703 effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the 704 presence of increasing concentrations of Fenofibrate, as detailed in A. C. Effect of 705 fenofibrate on the entry of luciferase expressing lentiviruses pseudotyped with SARS- 706 CoV-2 Spike in ACE2-expressing HEK-293T cells. Values are normalized to luciferase 707 expression by mock-treated cells set at 100%. Mean and s.e.m. from two experiments 708 with two replicates. Statistical deviations from 100% were assessed with a one sample t 709 test. D. Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS- 710 CoV-2 in the presence of increasing concentrations of MDL 28170, as detailed in A. E. 711 Comparison of MDL 28170 activity with entry inhibitors blocking viral endocytosis, 712 such as chloroquine and E-64d, and inhibitors blocking serine protease TMPRSS2, such 713 as camostat. ACE2 expressing HEK-293T cells transfected or not with TMPRSS2 were 714 exposed to SARS-CoV-2 Spike lentiviruses in the presence of these compounds. Values 715 are normalized to luciferase expression by mock-treated cells set at 100%. Mean and 716 s.e.m. from at least two experiments with two replicates. Statistical deviations from 717 100% were assessed with a one sample t test. 718 719 Figure 4. Decreased release of SARS-CoV-2 in the presence of inhibitors with 720 antiviral activity. A. Viral release to the supernatant in the presence of the indicated 721 compounds added at increasing concentrations 3 days post-infection of Vero E6 cells. 722 SARS-CoV-2 nucleoprotein was detected with an ELISA at concentrations were drugs 723 were nontoxic. Mean and s.e.m. from two experiments. B. Viral release to the 724 supernatant in the presence of the indicated interferons as described in A. Mean and 725 s.e.m. from one experiment. 726 727 SUPPLEMENTARY TABLES 728 Supp. Table 1. Antiviral activity of potential entry inhibitors tested against SARS-CoV-

729 2. IC50 values are reported in µM unless otherwise indicated. “Not active” refers to the 730 lack of inhibitory activity at the highest concentration tested for each compound. 23

731 Supp. Table 2. List of combinations of active antivirals that did not show any enhanced

732 antiviral activity when mixed. Synergy was defined as showing an enhanced IC50 in

733 drug combination as compared to the highest IC50 of the most potent compound tested. 734 Similar toxicity is reported if viability was found to be similar to the most toxic 735 compound tested in the combination, while higher toxicity reports an increase above 736 10% in cytotoxicity in the combination. 737 Supp. Table 3. Antiviral activity of potential post-entry inhibitors against SARS-CoV-

738 2. IC50 values are reported in µM unless otherwise indicated. “Not active” refers to the 739 lack of inhibitory activity at the highest concentration tested for each compound. 740 Supp. Table 4. Antiviral activity of potential inhibitors against SARS-CoV-2 with 741 predicted capacity to block SARS-CoV-2 viral protease. “Not active” refers to the lack 742 of inhibitory activity at the highest concentration tested for each compound. All 743 compounds but the two indicated were found non-toxic at 100 μM. 744 Supp. Table 5. Antiviral activity of potential inhibitors against SARS-CoV-2 with

745 unknown mechanism of action. IC50 values are reported in µM. “Not active” refers to 746 the lack of inhibitory activity at the highest concentration tested for each compound. 747 748 749 SUPPLEMENTARY FIGURES 750 Supplementary Figure 1. No antiviral activity of HIV-1 reverse transcriptase 751 inhibitors. Cytopathic effect on Vero E6 cells exposed to a fixed concentration of 752 SARS-CoV-2 in the presence of increasing concentrations of HIV-1 reverse 753 transcriptase inhibitors. Drugs were used at a concentration ranging from 0.0512 nM to 754 100 μM. Non-linear fit to a variable response curve from one experiment with two 755 replicates is shown (red lines). Cytotoxic effect on Vero E6 cells exposed to increasing 756 concentrations of drugs in the absence of virus is also shown (grey lines). 757 758 Supplementary Figure 2. Antiviral activity of NPO calpain and cathepsin inhibitors. 759 Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in 760 the presence of increasing concentrations of calpain and cathepsin inhibitors NPO. 761 MDL 28170 was used at a concentration ranging from 0.0512 nM to 100 μM, while 762 NPO was used from 0.00512 nM to 10 μM . Non-linear fit to a variable response curve 763 from one experiment with two replicates is shown (red lines). Cytotoxic effect on Vero

764 E6 cells exposed to increasing concentrations of drugs in the absence of virus is also 765 shown (grey lines). 766

767 AUTHOR CONTRIBUTION 768 Conceived and designed the experiments: JR, JMB, DPZ, JS, BC, JVA, NIU 769 Performed in silico drug modeling: VG 770 Performed experiments: JR, JMB, DPZ, MNJ, IE, VG, JVA, NIU 771 Contributed with critical reagents: CQ, IB 772 Analyzed and interpreted the data: JR, JMB, DPZ, MNJ, RP, LM, CQ, IE, IB, AV, VG, 773 JC, JB, JS, BC, JVA, NIU 774 Wrote the paper: JR, JVA, NIU 775 776 DATA AVAILABILITY Data is available from corresponding authors upon 777 reasonable request.

A Hydroxychloroquine Azithromycin Hydroxychloroquine & Azithromycin Virus = 2.1 µM = ~ 1.1 µM No virus 100 IC50 100 100 IC50 80 80 80 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055756; this version posted January 4, 2021. The copyright holder for this preprint

(% in RLUs ) 60 (which was not certified by peer review) is60 the author/funder, who has granted bioRxiv60 a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 40 40 40 20 20 20 0 0 Cytotoxicity 0

-6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log μM log μM log μM B 10 10 10 Virus Amantadine E-64d Methyl Beta Cyclodextrin NB-DNJ No virus 100 100 100 100 80 80 80 80 60 60 (% in RLUs ) 60 60 40 40 40 40 20 20 20 20 0 0 Cytotoxicity 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 -6 -4 -2 0 2 4 log μM log mM log10 μM 10 10 log10 μM Virus C 100 Camostat 100 AAT No virus 80 80 60 (% in RLUs ) 60 40 40 20 20

Cytotoxicity 0 0

-6 -4 -2 0 2 4 -2 -1 0 1 2 D log10 μM log10 mg/ml 140 120 100 P= 0.329 80 60 P= 0.0018 40 P= 0.0102 P= 0.025

% of viral entry (RLU s) 20 P= 0.0047 0

AAT Mock E-64d Vehicle NB-DNJ TofacitinibBaricitinib Camostat ChloroquineAmantadine CA-074-Me NiclosamideCiclesonideArbidol HCl 3'sialyllactose Chlorpromazine Mß-cyclodextrin HydroxychloroquineE 120 Mock Chloroquine 100 Camostat 80 60 P <0.0001 40 P <0.0001 20 % of viral entry (RLU s) 0 Figure 1 ACE2 ACE2 + TMPRSS2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055756; this version posted January 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A Remdesivir B Remdesivir & Hydroxychloroquine Virus Virus No virus No virus 100 100 = 80 IC50 1.2 µM 80 IC50= 1.2 µM 60 60 40 40 Cytotoxicity Citotoxicty (% in RLUs ) 20 (% in RLUs ) 20 0 0

-6 -4 -2 0 2 4 -6 -4 -2 0 2 4

log10 μM log10 μM

C Nelfinavir Mesylate Hydrate Lopinavir Tipranavir Amprenavir Darunavir Virus No virus 100 IC50= 1.4 µM 100 100 100 100 80 80 80 80 80 60 60 60 60 60 40 40 40 40 40 Cytotoxicity (% in RLUs ) 20 20 20 20 20 0 0 0 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log μM log μM 10 10 log10 μM log10 μM log10 μM

D Plitidepsin Plitidepsin & Hydroxychloroquine Plitidepsin & Remdesivir Virus No virus 80 80 80 IC50= 0.05 µM IC50= 0.01 µM IC50= 0.07 µM

60 60 60

40 40 40 Cytotoxicity (% in RLUs ) 20 20 20

0 0 0

-4 -3 -2 -1 0 -4 -3 -2 -1 0 -4 -3 -2 -1 0 log μM log10 μM 10 log10 μM Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055756; this version posted January 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A Virus B C Virus No virus 100 Itraconazole Fenofibrate No virus 80 100 100 IC50= 79.4 µM IC50= 20 µM 80 60 P= 0.0007 80

60 (RLU s) 60 40 40 % of viral entry Cytotoxicity

40 Cytotoxicity (% in RLUs ) (% in RLUs ) 20 20 20 0 0 0

-6 -4 -2 0 2 4 -6 -4 -2 0 2 4 Mock

log10 μM log10 μM Fenofibrate

D MDL 28170 Virus E No virus 200 = 0.2 µM ACE2 ACE2 + TMPRSS2 100 IC50 160 80 120 60 (RLU s) P= 0.0018 40 80 % of viral entry

Cytotoxicity P< 0.0001 (% in RLUs ) P< 0.0001 P= 0.0003 20 40 0 0 -6 -4 -2 0 2 4 Mock E-64d Mock E-64d log10 μM Camostat Camostat Chloroquine MDL 28170 Chloroquine MDL 28170

Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055756; this version posted January 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A Remdesivir B Hydroxychloroquine 2400 MDL 28170 600 INF alpha 1600 Nelfinavir mesylate hydrate INF gamma 800 Plitidepsin Fenofibrate 600 400 -2 Nucleocapsid (ng/ml )

400 -2 Nucleocapsid (ng/ml ) 200 200 SARS-Co V SARS-Co V

0 0

4 20 80 0.8 100 400 0.032 0.16 2.000 0.0064 10.000 0 00128 μM IU/ml Figure 4 Mode of Action IC / CC µM Previous Vendor DRUG 50 50 Mode of Action bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055756 (Mean +/-SD) ; this version posted January 4, 2021. TheClinical copyright Use holder for this Origenpreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-NDTargets 4.0 eukaryotic International Elongation license . nM Plitidepsin 0.06 +/- 0.02 / > 0.1 Multiple myeloma PharmaMar Factor 1A2 (eEF1A2)

MDL 28170 0.14 +/- 0.06 / > 87 Calpain III inhibitor & Cathepsin B Pre-Clinical Merck Landsteiner NPO-2142; -2143 & -2260 ~ 0.54 / >10 Calpain & Cathepsin Pre-Clinical inhibitors Genmed Remdesivir 2.16 +/-4.1 / > 85 RNA Polymerase inhibitor Ebola Virus Cayman Chemical

Clathrin-mediated endocitosys or Hydroxycloroquine 10.9 +/- 11.3 / > 96 Malaria Laboratorios Rubio pH-dependent viral fusion inhibitor Not calculated, < 25 µM Nelfinavir mesylate hydrate Protease inhibitor HIV-1 Sigma Aldrich but active < 10 / > 25 or Clathrin-mediated endocitosys or 2 Chloroquine 3.86 / > 25 Malaria Sigma Aldrich 10 IU/mL pH-dependent viral fusion inhibitor 2 2 Interferon 2 alpha 8.1+/-0.7 x10 IU/mL / >100 x10 IU/mL INF stimulated antiviral proteins Hepatitis & HIV-1 Sigma-Aldrich 2 2 Interferon gamma 11.2 x10 IU/mL / >100 x10 IU/mL INF stimulated antiviral proteins Granulomatous disease Sigma-Aldrich Fenofibrate 19.8+/- 8 / >100 Activates PPARα Dyslipidemia Lacer

Table 1 Tenofovir Emtricitabine Tenofovir & Emtricitabine Virus No virus 100 100 100 80 80 80 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055756; this version posted January 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license60 to display the preprint in perpetuity. It is made 60 available60 under aCC-BY-NC-ND 4.0 International license. 40 40 40 Cytotoxicity (% in RLUs ) 20 20 20 0 0 0

-6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log μM log10 μM log10 μM 10

TAF & Emtricitabine TAF 100 100 80 80 60 60 40 40 Cytotoxicity (% in RLUs ) Cytotoxicity (% in RLUs ) 20 20 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log10 μM log10 μM

Supplementary Figure 1 100 NP0-2142 100 NP0-2138 80 80 IC50= ~ 0.5 µM bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055756; this version posted January 4, 2021. The copyright holder for this preprint

(% in RLUs ) 60 60

(which was not certified by peer review) is the author/funder, who has granted(% in RLUs ) bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 40 40 20 20 Cytotoxicity 0 Cytotoxicity 0

-4 -3 -2 -1 0 1 2 -4 -3 -2 -1 0 1 2

log10 μM log10 μM

100 NP0-2260 100 NP0-2143 IC = 0.6 µM IC = ~ 0.4 µM 80 50 80 50 60 60 (% in RLUs ) (% in RLUs ) 40 40 20 20 Cytotoxicity 0 Cytotoxicity 0

-4 -3 -2 -1 0 1 2 -4 -3 -2 -1 0 1 2

log10 μM log10 μM Supplementary Figure 2 IC / CC µM Previous Vendor ACTIVITY DRUG 50 50 Mode of Action (Mean +/-SD) Clinical Use Origen

bioRxiv preprintHydroxychloroquine doi: https://doi.org/10.1101/2020.04.23.0557569.3 +/- 11.1 / > 80 ; this version posted January 4, 2021. The copyright holder for thisLaboratorios preprint Rubió ENTRY(which was not certified by peer review) is the author/funder, who hasClathrin-mediated granted bioRxiv endocitosys a license to ordisplay the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Malaria pH-dependent viral fusion inhibitor Chloroquine 3.9 / > 25 Sigma Aldrich

Not calculated, but Clathrin-mediated Amantadine Parkinson & influenza A Sigma Aldrich partialy active at 100 / > 100 endocitosys inhibitor Chlorpromazine Clathrin-mediated Not Active / > 18 Antipsychotic Sanofi (Largactil) endocitosys inhibitor CA-074-Me Not Active / > 34 Cathepsin inhibitor B Pre-Clinical Sigma Aldrich Not calculated, but E-64d Cathepsin inhibitor B/L Pre-Clinical Sigma Aldrich partialy active at 100 / > 100 Cholesterol-removing agent, Methyl-ß-cyclodextrin Not calculated, but Not approved Sigma Aldrich active at 1000 lipid raft disruption Not calculated, but Inhibits ceramide- glucosyltransferase Gaucher disease & NB-DNJ Calbiochem active at 100 / > 100 and β-glucosidase 2 Juvenile Sandhoff disease 3’ Sialyllactose Na Salt Not Active / 20 mM Inhibits viral binding Pre-Clinical Carbosynth Niclosamide Not Active / > 9 Beclin-1 stabilizer in autophagy Helmints Selleckchem

Ciclesonide Not Active / > 20 Glucocorticoid Asthma Selleckchem

Arbidol HCl Not Active / > 40 Fusion inhibitor? Influenza Selleckchem

Tofacitinib (Xeljanx) Not Active / > 100 JAK inhibitor Rheumatoid arthritis Pfizer Baricitinib Not Active / > 85 JAK inhibitor Rheumatoid arthritis Selleckchem

Camostat Not Active / > 100 TMPRSS2 inhibitor Chronic pancreatitis Merck Not calculated, but Cellular protease Alpha-1 Antitrypsin Alpha-1 antitrypsin deficiency Grifols active at 12.5 mg/ml / > 12.5 mg/ml inhibitor

Supplementary Table 1 Toxicity DRUG 1 DRUG 2 DRUG 3 DRUG 4 Synergy in combination

bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055756; this version posted January 4, 2021. The copyright holder for this preprint (which was not certified byAzithromycin peer review) is the author/funder, who has granted bioRxiv a license to displayNo the preprint in perpetuity.Similar It is made available under aCC-BY-NC-ND 4.0 International license. Lopinavir No Similar

Tipranavir No Similar

Hydroxy- Amprenavir No Similar Cloroquine Darunavir No Similar

Baricitinib No Similar

Tenofovir Emtricitabine No Similar

TAF Emtricitabine No Similar

Hydroxychloroquine No Similar

Amantadine No Similar

Remdesivir Chlorpromazine No Similar

Baricitinib No Higher

Tipranavir Lopinavir No Similar

Ritonavir No Higher

Tipranavir No Higher

Lopinavir Ritonavir Tenofovir Emtricitabine No Similar

Tenofovir Ritonavir Emtricitabine No Similar Alafenamide

Tipranavir Tenofovir Emtricitabine No Similar

Tenofovir Emtricitabine No Similar

Nelfinavir Hydroxychloroquine No Similar Mesylate Hydrate Remdesivir No Similar

Hydroxychloroquine No Similar

MDL28170 Remdesivir No Similar

Nelfinavir Mesylate Hydrate No Similar

Hydroxychloroquine No Similar

Remdesivir No Similar Plitidepsin MDL 28170 No Similar

Nelfinavir Mesylate Hydrate No Similar

Supplementary Table 2 22

IC / CC µM Previous Vendor ACTIVITY DRUG 50 50 Mode of Action (Mean +/-SD) Clinical Use Origen

RNA Polymerase inhibitor Ebola Virus Cayman Chemical bioRxivRemdesivir preprint doi: https://doi.org/10.1101/2020.04.23.0557562.2 +/-4.1 / > 85 ; this version posted January 4, 2021. The copyright holder for this preprint POST-ENTRY(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Galdesivir availableNot Active under / a100CC-BY-NC-ND 4.0RNA International Polymerase license inhibitor. YFV MedChemExpress Not calculated, but partially Flavivirus, Arenavirus, Favipiravir RNA polimerase inhibitor Quimigen active at 100 / > 100 Bunyavirus, Alphavirus Saquinavir Not Active / > 28 Protease inhibitor HIV-1 Reference standard HPLC Lopinavir Not calculated, but active at 20 / > 87 Protease inhibitor HIV-1 Abbot

RitonavirRitonavir NotNot Active active / 20-100/ 20-100 ProteaseProtease inhibitor inhibitor HIV-1 Abbot

Tipranavir Not calculated, but active at 20 / > 70 Protease inhibitor HIV-1 Reference standard HPLC

Nelfinavir Mesylate Hydrate Not calculated, but active <10 / > 25 Protease inhibitor HIV-1 Sigma Aldrich

Amprenavir Not calculated, but active at 100 / > 100 Protease inhibitor HIV-1 GSK

Fosamprenavir Calcium Not Active / > 25 Protease inhibitor HIV-1 Sigma Aldrich Not calculated, but partially active at 100 Darunavir Protease inhibitor HIV-1 Sigma Aldrich / > 100 Atazanavir Sulfate Not Active / > 20 Protease inhibitor HIV-1 Reference standard HPLC Tenofovir disoproxil fumarate Not Active / > 100 Reverse Transcriptase inhibitor HIV-1 Selleckchem Emtricitabine (Emtriva) Not Active / > 100 Reverse Transcriptase inhibitor HIV-1 Gilead Tenofovir Alafenamide Not Active / > 100 Reverse Transcriptase inhibitor HIV-1 Selleckchem

Velpatasvir Not Active / > 10 Iinhibitor of NS5A protein HCV Selleckchem

Sofosbuvir Not Active / > 100 Polymerase inhibitor HCV Selleckchem

Boceprevir Not Active / > 100 Protease inhibitor HCV Quimigen Vesatolimod Not Active / > 20 TLR7 agonist Hepatitis & HIV-1 MedChemExpress 2 2 Interferon 2 alpha 8.1+/-0.7 x10 IU/mL / >100 x10 IU/mL IFN stimulated antiviral proteins Hepatitis & HIV-1 Sigma-Aldrich 2 2 Interferon gamma 11.2 x10 IU/mL / >100 x10 IU/mL IFN stimulated antiviral proteins Granulomatous disease Sigma-Aldrich Targets eukaryotic Elongation Plitidepsin 0.06 +/- 0.02 / > 0.1 Multiple myeloma PharmaMar Factor 1A2 (eEF1A2)

Supplementary Table 3 11

Bioinformatic IC / CC µM Previous Vendor DRUG 50 50 Mode of Action ANALYSIS (Mean +/-SD) Clinical Use Origen bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055756; this version posted January 4, 2021. The copyright holder for this preprint (which wasSalbutamol not certified by peer review) is the Notauthor/funder, Active whoPredicted has granted SARS-Cov-2 bioRxiv Protease a license inhibitorto display the preprintAsthma in perpetuity. It is madeSigma Aldrich available under aCC-BY-NC-ND 4.0 International license. Diclondazolic acid (Lonidamine) Not Active Predicted SARS-Cov-2 Protease inhibitor Anticancer Abcam

Thiocolchicoside Not Active Predicted SARS-Cov-2 Protease inhibitor Skeletal muscle relaxant Sigma Aldrich Morphothiadine Not Active / 54 Predicted SARS-Cov-2 Protease inhibitor Hepatitis B Quimigen Glycogen phosphorylase Ingliforib Not Active Predicted SARS-Cov-2 Protease inhibitor Quimigen inhibitor Perampanel Not Active Predicted SARS-Cov-2 Protease inhibitor Antiepileptic Quimigen Thyrotropin-releasing Montirelin trifluoroacetate salt Not Active Predicted SARS-Cov-2 Protease inhibitor Sigma Aldrich hormone agonists Saquinavir Not Active Predicted SARS-Cov-2 Protease inhibitor HIV-1 Reference standard HPLC Pamicogrel Not Active / 6.4 Predicted SARS-Cov-2 Protease inhibitor Antiplatelet aggregation Quimigen Pomalidomide Not Active Predicted SARS-Cov-2 Protease inhibitor Anticonvulsants Sigma Aldrich Laflunimus Sodium Salt Not Active Predicted SARS-Cov-2 Protease inhibitor Anti-inflammatory Quimigen

Supplementary Table 4 Previous Vendor IC50 / CC50 µM ACTIVITY DRUG Mode of Action Clinical Use Origen bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055756 (Mean +/-SD) ; this version posted January 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Azithromycin (Zitromax) availableNot Active under / > a 100CC-BY-NC-ND 4.0 InternationalAntibiotic license. Bacteria Pfizer UNKNOWN Doxycycline (Anaclosil) Not Active / > 100 Antibiotic Bacteria Reig Tetraphase Eravacycline (Xerava) Not Active / > 4 Antibiotic Resistent bacteria Pharmaceuticals Quinacrine dihydrochloride Not ActiveNot Active / > 6 Inhibitor of NF-kappaB Parasites Sigma Aldrich Ivermectin (Stromectol) Not Active / > 2 Nuclear import inhibitor Parasites MSD Mefloquine hydrochloride Not Active / > 100 Phospholipid bilayer? Malaria Sigma Aldrich N-Acetil cystein (Flumil) Not Active / > 100 Synthesis of glutathione Influenza Zambon Inhibits OSBP, which produces the Itraconazole 79.37 / > 100 Fungus Janssen membrane-bound viral replication organelles Fluconazol Not Active / > 100 Antibiotic Fungus Franesius Kabi Famotidine Not Active/ > 100 Histamine-2 receptor antagonist Gastric Normon Cetirizine dihydorcloride Not Active/ > 100 Histamine-H1 receptor antagonist Antihistaminic Sigma Aldrich Colchicine Not Active / 0.63 Anti mytotic Gout attacks Merck Palbociclib Not Active/ 2,7 CDK4/6 inhibitor Breast cancer Selleckchem Ribociclib Not Active / > 20 CDK4/6 inhibitor Breast cancer Selleckchem Abenaciclib Not Active / > 1 CDK4/6 inhibitor Breast cancer Selleckchem Silibinin Not active / > 20 ? Liver disease Rottapharm Madaus

Atorvastatin Not active / > 20 HMG-CoA reductase inhibitor Cardiovascular disease Normon Fenofibrate 19.8 +/- 8 / > 100 Activates PPARα Dyslipidemia Lacer Calpain III inhibitor & MDL 28170 0.14 +/- 0.06 / > 87 Pre-Clinical Merck Cathepsin B inhibitor Landsteiner NPO-2142; -2143 & -2260 ~ 0.54 / > 10 Calpain & Cathepsin Pre-Clinical inhibitors Genmed Not calculated, but Calpain & Cathepsin Landsteiner NPO-2138 Pre-Clinical partially active at 100 / > 10 inhibitors Genmed

Supplementary Table 5