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Biomedicine & Pharmacotherapy 164 (2023) 114997 Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha β-Cyclodextrins as affordable antivirals to treat coronavirus infection Dalia Raïch-Regué a, 1, Raquel Tenorio b, 1, Isabel Fernández de Castro b, 1, Ferran Tarrés-Freixas c, d, 2, Martin Sachse b, 2, 3, Daniel Perez-Zsolt a, 2, Jordana Muñoz-Basagoiti a, 2, Sara Y. Fernández-Sánchez b, Marçal Gallemí a, Paula Ortega-González b, 4, Alberto Fernández-Oliva b, 5, José A. Gabaldón e, Estrella Nuñez-Delicado e, Josefina Casas f, g, Núria Roca c, d, Guillermo Cantero c, d, Mónica Pérez c, d, Carla Usai c, d, Cristina Lorca-Oró c, d, Júlia-Vergara Alert c, d, Joaquim Segalés d, h, Jorge Carrillo a, Julià Blanco a, i, j, Bonaventura Clotet Sala a, i, j, José P. Cerón-Carrasco k, *, Nuria Izquierdo-Useros a, j, **, Cristina Risco b, *** a IrsiCaixa, Germans Trias i Pujol Research Institute (IGTP), Can Ruti Campus, 08916, Badalona, Spain b Cell Structure Lab, Centro Nacional de Biotecnologia, CNB - CSIC, Campus de Cantoblanco, 28049 Madrid, Spain c IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la UAB, 08193 Bellaterra (Cerdanyola del Vallès), Spain d Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Barcelona, Spain e Reconocimiento y Encapsulación Molecular. Universidad Cató lica San Antonio de Murcia (UCAM), Campus de los Jeró nimos, N◦ 135, Guadalupe, 30107 Murcia, Spain f Institut de Química Avançada de Catalunya (IQAC-CSIC), Barcelona, Spain g Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Instituto de Salud Carlos III, 28029 Madrid, Spain h Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, 08193 Bellaterra (Cerdanyola del Vallès), Spain i University of Vic–Central University of Catalonia (UVic-UCC), 08500 Vic, Spain j Consorcio Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, 28029 Madrid, Spain k Centro Universitario de la Defensa, Universidad Politécnica de Cartagena, C/Coronel Ló pez Peña s/n, Base Aérea de San Javier, Santiago de la Ribera, 30720 Murcia, Spain A R T I C L E I N F O A B S T R A C T Keywords: The SARS-CoV-2 pandemic made evident that there are only a few drugs against coronavirus. Here we aimed to SARS-CoV-2 identify a cost-effective antiviral with broad spectrum activity and high safety profile. Starting from a list of 116 COVID-19 drug candidates, we used molecular modelling tools to rank the 44 most promising inhibitors. Next, we tested Coronavirus their efficacy as antivirals against α and β coronaviruses, such as the HCoV-229E and SARS-CoV-2 variants. Four Antiviral Drug repurposing drugs, OSW-1, U18666A, hydroxypropyl-β-cyclodextrin (HβCD) and phytol, showed in vitro antiviral activity Cyclodextrin against HCoV-229E and SARS-CoV-2. The mechanism of action of these compounds was studied by transmission β-cyclodextrin electron microscopy and by fusion assays measuring SARS-CoV-2 pseudoviral entry into target cells. Entry was inhibited by HβCD and U18666A, yet only HβCD inhibited SARS-CoV-2 replication in the pulmonary Calu-3 cells. Compared to the other cyclodextrins, β-cyclodextrins were the most potent inhibitors, which interfered with viral fusion via cholesterol depletion. β-cyclodextrins also prevented infection in a human nasal epithelium model ex vivo and had a prophylactic effect in the nasal epithelium of hamsters in vivo. All accumulated data point to * Corresponding author at: Centro Universitario de la Defensa, Universidad Politécnica de Cartagena, C/Coronel Ló pez Peña s/n, Base Aérea de San Javier, Santiago de la Ribera, 30720 Murcia, Spain. ** Corresponding author at: IrsiCaixa, Germans Trias i Pujol Research Institute (IGTP), Can Ruti Campus, 08916, Badalona, Spain. *** Corresponding author at: Cell Structure Lab, Centro Nacional de Biotecnologia, CNB - CSIC, Campus de Cantoblanco, 28049 Madrid, Spain. E-mail addresses: jose.ceron@cud.upct.es (J.P. Cerón-Carrasco), nizquierdo@irsicaixa.es (N. Izquierdo-Useros), crisco@cnb.csic.es (C. Risco). 1 Equal contribution as first authors 2 Equal contribution as second authors 3 Present address: Centro Nacional de Microbiología/ISCIII, Majadahonda, Madrid, Spain. 4 Present address: CBGP, UPM-INIA, Madrid, Spain. 5 Present address: Alcura Health España & AmeriSourceBerguen, Barcelona, Spain. https://doi.org/10.1016/j.biopha.2023.114997 Received 13 January 2023; Received in revised form 1 June 2023; Accepted 6 June 2023 Available online 8 June 2023 0753-3322/© 2023 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 β-cyclodextrins as promising broad-spectrum antivirals against different SARS-CoV-2 variants and distant alphacoronaviruses. Given the wide use of β-cyclodextrins for drug encapsulation and their high safety profile in humans, our results support their clinical testing as prophylactic antivirals. 1. Introduction two representative viruses from the α and β coronavirus genera: the HCoV-229E and SARS-CoV-2 viruses. With a combination of computa­ Since the start of the SARS-CoV-2 pandemic novel vaccines have tional chemistry, virology, cell biology and electron microscopy been developed that have changed the global landscape protecting from methods, we studied 44 compounds. Four of them showed antiviral severe coronavirus disease (COVID-19) and decreasing the death toll activity against HCoV-229E and SARS-CoV-2, β-cyclodextrins being the during the early stages of the pandemic. Yet, it has taken more than a most promising candidates to treat the infection caused by SARS-CoV-2. year for COVID-19 vaccines to reach 20 % of the population in low- income nations. Even in wealthy countries there is only about an 80 % 2. Material and methods of vaccination rate (https://ourworldindata.org/covid-vaccinations). Post- vaccination, vulnerable and immunocompromised individuals are 2.1. Molecular modelling still at high risk upon infection [2]. Unfortunately, all vaccines approved so far have failed to confer sterilizing immunity, and may not fully 2.1.1. Viral targets protect from infection. The characterization of the crystal structure of the Mpro [25], an Under this scenario, therapeutic approaches are still needed to pro­ enzyme recruited by the SARS-CoV-2 to complete the replication and tect those at higher risk after SARS-CoV-2 infection, which include in­ transcription steps, was included in the protein data bank (PDB) library dividuals who lack access to vaccine regimens or those who are (code 6LU7) and allowed the use of virtual screening (VS) techniques vaccinated but fail to mount adequate immune responses. Safe, afford­ [3]. Indeed, the same authors performed VS simulation searching for able and effective antivirals could be of great help to mitigate these antiviral drugs in an in-house library of compounds [25]. Many other contingencies, offering treatment for those individuals not only at structures have been resolved since then, and there are several PDB higher risk of developing severe COVID-19, but also for those with entries with inhibitors located in the active site of the Mpro target (i.e. moderate or mild outcomes that could also benefit from reducing the codes 5RG1, 5RGL, 7A1U, 7JU7) [10,17]. Most of techniques aim for time and disease-associated symptoms. WHO recommends different small molecules to reach the active site, which is characterized by a treatments that limit severe disease or risk of hospitalization [1]. Some catalytic dyad of Cys145 and His41 residues [61]. of these antiviral treatments are based on neutralizing antibodies or The SARS-CoV-2 spike has been also extensively assessed through VS intravenously administered drugs that are dispensed in the hospitals simulations as it governs host attachment and virus–cell membrane only. Therapeutic antibodies such as casirivimab-imdevimab have fusion upon infection [58]. In that framework, the characterization of shown activity in clinical trials before the surge of new variants. How­ the receptor-binding domain (RBD), deposited with PDB code 6M0J by ever, pre-clinical studies suggest that this combination lacks neutrali­ Wang et al., 2020, allows for developing inhibitors by using molecular zation activity against omicron [1]. In the case of sotrovimab, although models [51]. It has been also proposed that the SARS-CoV-2 spike might activity might be retained, higher concentrations of the antibodies bind two metabolites (biliverdin and bilirubin) avoiding the antibody would be needed for neutralization of omicron [1]. Remdesivir targets recognition [43]. the viral RNA polymerase but still requires intravenous treatment, Within the last two years extensive research have led the discovery of although new orally-available formulations are about to be tested [44]. other X-ray structures, for instance papain-like protease (PLpro), an Molnupiravir, another inhibitor of the viral RNA polymerase, is enzyme that regulates SARS-CoV-2 viral spread and innate immunity administered orally and like remdesivir has shown clinical benefits [48]. Some of the more recent crystals with non-covalent inhibitors are when administered early upon infection [23]. WHO weakly recom­ associated to the PDB codes 7CMD, 7JIR, 7JIT, 7JIV, 7JIW, 7JN2, 7JRN, mends administration of Molnupiravir, and only for patients at high risk 7KOJ, 7KOK, 7KOL, 7KRX, 7LBR, 7LBS, 7LLF, 7LLZ and 7LOS [13,33,34, of being admitted to hospital with COVID-19 [1]. Paxlovid is taken 40,47]. In addition, the non-structural protein 16 (NSP16) has been orally, but is not widely accessible and imply costs that exceed those of shown to play an essential role for immune evasion by mimicking the vaccines [7]. Moreover, Paxlovid is only available for patients that human homolog, CMTr1. However, unlike CMTr1, NSP16 needs to form tolerate Ritonavir as part of the treatment with the antiviral nirma­ a heterodimer with NSP10 to activate its enzymatic activity. Although trelvir, and has also raised concerns as monotherapy due to associated there are no available crystals for the inactive monomeric NSP16, viral resistance [59], which may limit efficacy against new variants of Bowman and co-workers determined its activation mechanism and the concern (VOC). location of a cryptic pocket [57], which if targeted with a small mole­ None of the antivirals currently approved have the cost-effective cule, can be used to inhibit NSP16. profile or the easy administration route needed to offer prophylaxis to Niemann-Pick type C1 (NPC1), a lipid-transfer protein that regulates those vulnerable individuals at higher risk of developing severe disease intracellular cholesterol traffic, has been shown to play a role in human upon SARS-CoV-2 infection on a global scale. Nevertheless, the key to cell infection [14]. Within that hypothesis, the regulation of cholesterol control the SARS-CoV-2 pandemic might follow an approach similar to by targeting NPC1 might offer an additional therapeutic strategy to treat HIV-1 pre-exposure prophylaxis (PrEP), which has been employed to infected patients. The experimental data for NPC1 is still scarce, with decrease the HIV-1 infection rates [32]. With the success of this prior only two X-ray structures available (PDB codes 6UOX and 5U73). HIV-1 strategy, having an affordable antiviral with prophylactic po­ tential and a broad-spectrum activity at the initial surges of novel VOCs 2.1.2. Library preparation, protein refinement and virtual screening could be key to decrease SARS-CoV-2 transmission rates. Our in-house library of 116 compounds were prepared for simula­ Here we aimed to identify a cost-effective antiviral with broad tions by using LigPrep (Schrödinger Release 2021–3). Initial cartesian spectrum activity and high safety and tolerability profiles. We began coordinates were retrieved from the PubChem database (Kim et al., compiling a list of drugs previously used to treat other pathologies or 2021). Geometries were subsequently optimized by assessing proton­ characterized in preclinical studies with potential to treat coronavirus ation and tautomeric states at pH 7 ± 2 as predicted by Epik [15,46]. All infections. We next employed molecular modelling tools to rank the other parameters are set as default as implemented in the Schrödinger most promising inhibitors and tested their efficacy as antivirals against suite of programs (Schrödinger Release 2021–3). All abovementioned 2 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 Table 1 List of in vitro tested compounds against HCoV229E. Num. Drug IC50 ± SD/ Mode of action Previous clinical use Vendor CC50 ± SD Origin A 1 OSW-1 0.5 ± 0.1 nM / OSBP inhibitor Pre-clinical Cayman > 100 nM Chemical 2 HβCD 4.3 ± 4.0 / 43.6 Extracts cholesterol from cellular membranes Vaccine adjuvant Drug carrier Trappsol ± 5.7 mM Cyclo 3 U18666A 2.6 ± 1.1 / 74.4 NPC1 inhibitor Pre-clinical Millipore ± 14.8 μM 4 Phytol 18.6 ± 6.9 / Activator of the PPARs pathway and inhibitor of NF-κB Pre-clinical Sigma Aldrich 56.2 ± 7.7 μM B 5 Mdivi-1 11.7 ± 0.7 / Inhibitor of Drp1-dependent mitochondrial fission and Pre-clinical Sigma Aldrich 95.4 ± 1.8 µM Bax/Bak-dependent cytochrome c release during apoptosis 6 FLI 06 0.4 ± 0.3 / > Inhibitor of Notch signaling Pre-clinical Sigma Aldrich 50 μM 7 Gingenoside RB1 48.3 ± 2.1 / > Reversible inhibitor of PIK93γ and PI4KIIIβ Pre-clinical Sigma Aldrich 1000 μM 8 Digitoxin 0.021 ± 3.1 / Inhibitor of the Na-K-ATPase membrane pump Cystic fibrosis Sigma Aldrich 6.9 ± 6.3 nM 9 PIK93 4.2 ± 1.4 / > Reversible inhibitor of PIK93γ and PI4KIIIβ Pre-clinical Sigma Aldrich 50 μM 10 Bortezomib (PS- 0.6 ± 0.3 / > Inhibitor of proteasome Multiple myeloma Mantle cell lymphoma Selleckchem 341) 100 nM 11 Aspirin 1.0 ± 9.8 / 1.1 Inhibitor of prostaglandin synthesis and platelet Pain Fever Inflammation Sigma Aldrich ± 1.7 mM aggregation 12 Wortmannin 21.6 ± 15.6 / > Inhibitor of PI3Ks Pre-clinical Millipore 50 μM 13 Alpha-terpineol 381.7 ± 107.1 / Inhibitor of NF-κB activity, COX-2 and IKK2 Adjuvant in treatment for upper respiratory Sigma Aldrich 1058.6 ± tract congestion 332.6 μM 14 Dehydrocostus 4.2 ± 1.8 / 39.1 Inhibitor of the IKKβ/NF-κB/COX-2 and PI3K/Akt/Bad Pre-clinical Sigma Aldrich lactone ± 3.7 uM signaling pathways 15 Valinomycin 2.5 ± 0.2 / 12.5 Binds and exchanges potassium across membranes Pre-clinical Sigma Aldrich ± 3.5 μM 16 Terpinolene 8 ± 2.2 mM/ 69 ND Pre-clinical Sigma- ± 5 mM Aldrich C 17 Baclogen NA / > 1000 GABA-B receptor agonist activity Multiple sclerosis Hepatocellular carcinoma Sigma Aldrich μM Liver cirrhosis 18 CI976 NA / 50.7 ± Inhibitor of sterol O-acyltransferase and acyl-coenzyme Pre-clinical Sigma Aldrich 12.1 μM A: cholesterol acyltransferase. Inhibitor of Golgi- associated LPL acyltransferase (LPAT) 19 Sulfacetamide NA / > 1 mM Inhibitor of the dihydropteroate synthase. Inbibitor of Antibiotic Sigma Aldrich bacterial para-aminobenzoic acid 20 Squalene NA / 135.4 ± Intermediate metabolite in the synthesis of cholesterol Pre-clinical Sigma Aldrich 5.4 mM 21 Sodium 4- NA / > 500 mM Interacts with hydrophobic regions in unfolded proteins Urea cycle disorders Sigma Aldrich phenylbutyrate and reduces ER stress 22 Oleanolic acid NA / 25.2 ± ND Pre-clinical Sigma Aldrich 7.4 mM 23 MitoTEMPO NA / NA Mimetic of the mitochondria-targeted superoxide Pre-clinical Sigma Aldrich dismutase and scavenger of the superoxide and alkyl radicals 24 N-acetyl-L-cystein NA / NA Reductant of disulfide bonds and scavenger of reactive Mucolytic agent Chronic and acute Sigma Aldrich oxygen species bronchitis Pulmonary fibrosis and emphysema Acetaminophen intoxication 25 Camphene NA / 72 ± 9.2 Reduces plasma cholesterol and triglycerides Pre-clinical Sigma Aldrich mM 26 Myricetin NA / 433.6 ± Inhibitor of the cyclooxygenase 1 Pre-clinical Sigma- 214.1 μM Aldrich 27 Aminohipurric NA / < 10 mM ND Test of renal plasma flow Sigma- Aldrich 28 L-Phenilalanine NA / < 10 mM ND Depression Vitiligo Sigma- Aldrich 29 (-)β-Pinene NA / 293.6 ± Inhibitor of acetylcholinesterase and positive modulator Treatment for bladder, kidney, and urinary Sigma Aldrich 103.8 μM of GABA receptors stones 30 R-(+)-Limonene NA / 1908.4 ± Angiogenesis inhibitor and TNF and IL-6 suppressor Genital Human Papilloma Virus infection HWI group 194.2 μM Parotid Gland Tumor Submandibular Gland Tumor 31 (-)-Trans- NA / 1835 ± Agonist of the CB2, inhibitor of p38 MAPK/NF-κB and Clinical trial for analgesia Sigma Aldrich Caryophyllene 173 μM reductor of the cytokine release 32 Esential Oil Pinus NA / 533.3 ± Down-regulator of Cdc25C and cell cycle. Enhancer of Pre-clinical Terpenic sylvestris 57.7 μg/mL* calcium channels activation 33 Hydrosol Pinus NA / > 10 mg/ ND Pre-clinical Le Gattilier sylvestris mL* The Table is divided in three sections: 1) Compounds with antiviral activity against HCoV229E and SARS-CoV-2 (A-white, 1–4); 2) compounds with antiviral activity against HCoV229E but not against SARS-CoV-2 (B-light grey, 5–16) and 3) compounds without antiviral activity against HCoV229E (C-dark grey, 17–33). For each 3 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 compound, IC50 and CC50 values for HCoV229E, known mode of action, previous clinical use and vendor origin are indicated. ND, non-determined. *These are complex mixtures of compounds (molarity unknown) PDB structures were refined with the Protein Preparation Wizard mod­ activity of living cells [45,54]. MRC-5 cells were cultured in a 96-well ule [30], a multistep protocol. During that structural refinement, plate until 80 % confluency was reached. Serial dilutions of the com­ missing hydrogen atoms are added to the protein, charges were pounds in DMEM with 10 % FBS were added in triplicate and in a final assigned, protonation states were determined with Epik at pH 7.0 ± 2.0, volume of 100 μL/well. After 24 h, 5 mg/mL of MTT reagent (Sig­ and a final restricted minimization is performed with the OPLS4 force ma-Aldrich) was added to a final concentration of 0.5 mg/mL. Incuba­ field [29]. A grid was created by considering co-crystal ligand or refer­ tion was maintained during 4 h at 37ºC with 5 % CO2 for metabolization ence residues, as in the case of allosteric sites. Two docking runs were of the reagent and then 100 μL of lysis buffer (10 % SDS, 0.01 M HCl and carried out, with single-precision (SP) or extra-precision (XP) scoring 85 % isopropanol) was added and incubation maintained for 30 min on functions [12,20]. For the records, ten poses per ligand were saved for an orbital shaker protected from light. Plates were read at 570 nm and each docking run. Binding energies are eventually computed with the the background (measured at 690 nm) was subtracted. Data ware molecular mechanics/generalized Born surface area (MMGBSA) imple­ calculated from three independent replicates. mented in Prime module [21,22], a more refined method that accounts for the energies before and after ligand binding [39,60]. The resulting 2.2.3. Antiviral activity free energies (ΔG) are therefore used for ranking compounds of our li­ The effect of the compounds on viral infection was determined by IF brary. The last computational step uses molecular dynamics (MD) to as follows: 20,000 MRC5 cells/well were seeded in duplicates per drug assess the dynamic stability of the generated target-drug adducts. An and condition in 96-wells flat-bottom plates. When 80 % confluency was orthorhombic box of TIP3P-waters was generated by using periodic reached, cells were adsorbed with HCoV-229E in DMEM media without boundary conditions with a buffer distance of 10 Å. Sodium cations are FBS at MOI= 0.1 PFU/cell, and incubated for 1 h at 37 ◦ C. The inoculum added to keep the system electronically neutral while additional ions was then removed and a serial dilution of the compounds in DMEM were incorporated to mimic the physiological salt concentration of 0.15 supplemented with 1 % FBS was added. A duplicate of the infected cells M NaCl. The MD protocol includes minimization as implemented by without any drug treatment served as control. After 24 h, cells were default in the GPU-accelerated version of Desmond [5]. The MD simu­ fixed with 4 % PFA for 20 min and washed three times with PBS. Cells lation is completed by producing trajectories of 100 ns. were permeabilized with 0.25 % saponin in PBS for 10 min and then During our investigation several publications showed the antiviral treated 30 min with blocking buffer (1xPBS with 0.25 % saponin and 2 activity of 10 compounds against SARS-CoV-2 (Simvastatin, Sirolimus, % FBS). Cells were then incubated 1 h with a rabbit antibody specific for Everolimus, Itraconazole, Quercetin, Taxifolin, Resveratrol, Digoxin, the HCoV-229E nucleocapsid (N) protein (Ingenasa) diluted 1:200 in Lanatoside C and SilibininA) that we found to inhibit 229E virus, and blocking buffer. After 3 washes with PBS, cells were incubated 45 min those were excluded from our study due to the lack of novelty [9,24,27, with an anti-rabbit secondary antibody conjugated with Alexa fluor 488 42,55]. Indeed, some of them (Simvastatin, Sirolimus, Itraconazole, (Invitrogen) diluted 1:500 in blocking buffer and washed three times Quercetin, Resveratrol and Silibinin A) have been or are currently with PBS. Finally, cell nuclei were labeled 20 min with 4′ ,6-diamidino-2- included in clinical trials for COVID-19 (https://clinicaltrials.gov). phenylindole (DAPI) diluted 1:200 in blocking buffer and cells then washed 3 times with PBS. Images were obtained with a Leica DMi8 S 2.1.3. Reagents used widefield epifluorescence microscope and processed with Image J soft­ All the compounds and respective vendor origin are detailed in ware. Data were normalized by setting the positive infection control as Table 1, and Table S1. 100 % of infection. Inhibition data were plotted as dose-effect curves fitted to a nonlinear regression model in GraphPad Prism v 9.4 software. 2.2. Assays with HCoV-229E The IC50 was calculated with Quest Graph™ IC50 Calculator (https:// www.aatbio.com/tools/ic50-calculator). All experiments were repli­ 2.2.1. Cells and virus cated three times. MRC5 cells (ATCC CCL-171) were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10 % inactivated fetal 2.3. Antiviral activity in cells infected with SARS-CoV-2 bovine serum (FBS, Biological Industries), 4 mM glutamine (Sigma- Aldrich), 1 x non-essential amino acid solution (Sigma-Aldrich), 100 U/ 2.3.1. Biosafety statement mL penicillin and 100 μg/mL streptomycin (both from Sigma-Aldrich). The biosafety committee of the Institute Germans Trias i Pujol For preparing virus stocks, human coronavirus 229E (HCoV-229E; approved the execution of SARS-CoV-2 experiments at the BSL3 labo­ ATCC VR-740) was propagated in MRC-5 cells as described [31] with a ratory of the Center for Bioimaging and Comparative Medicine (CSB- modification that is maintaining cell cultures at 35ºC. Viral titer was 20–015-M7). calculated as 50 % tissue culture infective dose (TCID50). Briefly, MRC-5 cells in a 96-well plate at 80 % confluency were inoculated with a serial 2.3.2. Cells dilution of the viral stock, from 10-1 to 10-8. The plate was incubated at Vero E6 cells (ATCC CRL-1586) were cultured in Dulbecco’s modi­ 33 ◦ C and with 5 % CO2 for 5 days and then fixed with 4 % para­ fied Eagle medium, (DMEM) supplemented with 10 % fetal bovine formaldehyde (PFA) at room temperature (RT) for 20 min. Monolayers serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM were processed by indirect immunofluorescence (IF) using specific an­ glutamine (all from Invitrogen). HEK-293 T cells (ATCC repository) tibodies against the HCoV-229E nucleocapsid (N) protein (see below), were maintained in DMEM with 10 % FBS, 100 IU/mL penicillin and and titer calculated as described [38,41]. 100 μg/mL streptomycin (all from Invitrogen). HEK-293 T cells over­ expressing the human ACE2 (293 T-ACE2) were kindly provided by 2.2.2. Cytotoxicity assay Integral Molecular Company and maintained in DMEM with 10 % FBS, A stock solution of each compound was prepared. Hydrophobic 100 IU/mL penicillin and 100 μg/mL streptomycin, and 1 μg/mL of compounds were dissolved in dimethyl sulfoxide (DMSO) or ethanol (as puromycin (Invitrogen). CaLu-3 cells were kindly provided by the lab­ recommended by the manufacturer) and stored at − 20 ◦ C. To evaluate oratory of Dr. Sanchez Cespedes and maintained in DMEM with 10 % the viability of cell cultures when treated with the compounds, we FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin. performed an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazo­ lium bromide) assay, which measures the mitochondrial dehydrogenase 4 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 2.3.3. Virus isolation and sequencing luminometer at the time of supernatant collection. SARS-CoV-2 variants were isolated from clinical nasopharyngeal swabs in Vero E6 cells, as previously described [42]. Viral stocks were 2.3.7. IC50 calculation and statistical analysis grown in Vero E6 cells and supernatants were collected and stored at Response curves of compounds or their mixes were adjusted to a non- − 80ºC until use. The following SARS-CoV-2 variants with deposited linear fit regression model, calculated with a four-parameter logistic genomic sequence at the GISAID repository (http://gisaid.org) were curve with variable slope. Cells not exposed to the virus were used as tested: B.1 (D614G) isolated in Spain in March 2020 (EPI_ISL_510689); negative controls of infection and were set as 100 % of viability to and 4 variants isolated in Spain from January to February 2021: Alpha normalize data and calculate the percentage of cytopathic effect. All or B.1.1.7 (EPI_ISL_1663569), β or B.1.351 (originally detected in South analyses and Figures were generated with the GraphPad Prism v8.0b Africa; EPI_ISL_1663571), Zeta or P.2 (originally detected in Brazil; Software, Adobe Photoshop or BioRender. EPI_ISL_1831696), Delta or B.1.617.2 (originally detected in India; EPI_ISL_3342900), and Omicron or B.1.1.529 (EPI_ISL_8151031). 2.4. Transmission electron microscopy (TEM) Genomic sequencing was performed from viral supernatant by using standard ARTIC v3 or v4 based protocols followed by Illumina Mock- or SARS-CoV-2 – infected (MOI = 0.02 PFU/cell) cell mono­ sequencing [dx.doi.org/10.17504/protocols.io.bhjgj4jw]. Raw data layers were incubated in the absence or presence of two different con­ analysis was performed by viralrecon pipeline [https://github. centrations of the compounds and fixed at 48 hpi with 4 % com/nf-core/viralrecon] while consensus sequence was called using paraformaldehyde and 1 % glutaraldehyde in phosphate buffered saline samtools/ivar at the 75 % frequency threshold. Viral variants were (PBS) for 2 h at room temperature (RT). Cells were removed from the titrated at 1/3 dilutions on Vero E6 cells using the same luminometric plates in the fixative, pelleted by centrifugation and washed three times assay described for antiviral testing. Thus, for all VOCs, we used with PBS. Post-fixation of cell pellets was done on ice with 1 % osmium equivalent infectious units inducing 50 % of viral induced cytopathic tetroxide + 0.8 % potassium ferrocyanide in water. Afterwards, pellets effect. were dehydrated on ice with increasing concentrations of acetone and processed for embedding in the epoxy resin EML-812 (Taab Labora­ 2.3.4. Pseudovirus production tories), as previously described [52]. Infiltration with epoxy resin was HIV-1 reporter pseudovirus expressing SARS-CoV-2 Spike protein performed at RT. All samples were polymerized at 60 ◦ C for 48 h. Ul­ and luciferase were generated using two plasmids. pNL4–3. Luc.R-.E- trathin sections (50–70 nm) were cut with a Leica UC6 microtome and was obtained from the NIH AIDS repository. SARS-CoV-2. SctΔ19 was placed on uncoated 300 mesh copper grids. Sections were contrasted generated (Geneart) from the full protein sequence of SARS-CoV-2 spike with 4 % uranyl acetate and Reynold’s lead citrate. Images were taken with a deletion of the last 19 amino acids in C-terminal, human-codon with a Tecnai G2 TEM operated at 120 kV with a Ceta camera or with a optimized and inserted into pcDNA3.4-TOPO [35]. Spike plasmid was Jeol 1400 operated at 120 kV with a Gatan Rio camera. At least 100 cells transfected with X-tremeGENE HP Transfection Reagent (Merck) into per condition were studied by TEM. HEK-293 T cells, and 24 h post-transfection, cells were transfected with pNL4–3. Luc.R-.E-. Supernatants were harvested 48 h later, filtered with 2.5. SARS-CoV-2 Mpro inhibition assay 0.45 μM (Millex Millipore) and stored at − 80ºC until use. The p24gag content of all viruses was quantified using an ELISA (Perkin Elmer) and A SARS-CoV-2 3CL main protease (MBP-tagged) assay kit (BPS viruses were titrated in HEK-293 T overexpressing the human ACE2. Bioscience) was used following the kit instructions. In brief, the assay buffer was prepared by adding dithiothreitol, and then the protease was 2.3.5. Pseudoviral entry inhibition assay diluted to a 3–5 ng/μL. Next, diluted protease solution was added to the HEK-293 T overexpressing the human ACE2 were used to test the test samples and the positive controls. The MβCD and the GC376 control indicated compounds. A constant pseudoviral titer was used to pulse solutions were prepared with the assay buffer. The reaction was started cells in the presence of the drugs. At 48 h post-inoculation, cells were by adding the substrate solution to each well and incubated overnight. lysed with the Bright Glo Luciferase system (Promega). Luminescence Fluorescence was read in an EnSight multimode plate reader (Perkin was measured with an EnSight Multimode Plate Reader (Perkin Elmer). Elmer) at a 360 nm/460 nm excitation/ detection wavelength. Per­ centage of Mpro activity inhibition is calculated as follows: (positive 2.3.6. Antiviral activity against SARS-CoV-2 control – test inhibitor) / positive control. Increasing concentrations of the indicated antiviral compounds were added to Vero E6 cells and immediately after, we added equivalent in­ 2.6. Lipidomic analysis of plasma membranes of Calu-3 cells after MβCD fectious units of SARS-CoV-2 variants that induced a 50 % cytopathic treatment effect. Untreated non-infected cells and untreated virus-infected cells were used as negative and positive controls of infection, respectively. To To assess the lipid composition, 3 million Calu-3 cells/well were detect any drug-associated cytotoxic effect, Vero E6 cells were equally seeded in a 6-well plate and treated with 0, 0.6 or 2.5 mM of MβCD for 2 cultured in the presence of increasing drug concentrations, but in the h at 37 ºC and 5 % CO2. After extensive washing with PBS, cells were absence of virus. Cytopathic or cytotoxic effects of the virus or drugs detached with trypsin and stored at − 80 ºC until its analysis. A total of were measured 3 days after infection, using the CellTiter-Glo lumines­ 750 μL of a methanol-chloroform (1:2, vol/vol) solution containing in­ cent cell viability assay (Promega). Luminescence was measured in a ternal standards (16:0 D31_18:1 phosphocholine, C17:0 cholesteryl Fluoroskan Ascent FL luminometer (ThermoFisher Scientific). ester, stigmasterol and N-dodecanoylsphingosylphosphorylcholine, 0.2 Viral replication of SARS-CoV-2 was also assessed on Calu-3 cells in nmol each, from Avanti Polar Lipids) were added to 300,000 cells. the presence of the indicated antiviral compounds. Compounds were Samples were vortexed and sonicated until they appeared dispersed and incubated with cells before adding the SARS-CoV-2 virus at MOI = 0.3. extracted at 48 ◦ C overnight. The samples were then evaporated and After 24 h of incubation at 37ºC and 5 % CO2, cells were washed with transferred to 1.5 mL Eppendorf tubes after the addition of 0.5 mL of PBS and compounds were added in fresh media at the same concentra­ methanol. Samples were evaporated to dryness and stored at − 80 ◦ C tion for 48 h. The amount of SARS-CoV-2 nucleoprotein released to the until analysis. Before analysis, 150 μL of methanol were added to the supernatant was measured with SARS-CoV-2 nucleocapsid protein High- samples, centrifuged at 13,000 g for 3 min, and 130 μL of the superna­ Sensitivity Quantitative ELISA (ImmunoDiagnostics) according to the tants was transferred to ultra-performance liquid chromatography manufacturer’s protocol. The cytopathic effect on Calu-3 cells was (UPLC) vials for injection and analysis with an Acquity UPLC system assessed with Cell Titer-Glo Assay with a Fluoroskan Ascent FL (Waters) connected to a time-of-flight (TOF; LCT Premier XE, Waters) 5 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 Fig. 1. Virtual screening and molecular dynamics for selecting antiviral drugs. (A) Schematic representation of our library of compounds; experimental structures for all selected viral targets displayed in cartoons. (B) Chemical structures for the best-ranked compounds. (C) Poses adopted by the best-ranked com­ pounds (targets are sketched in grey cartoons and surface); the associated root mean-square deviation (RMSD) in angstroms (Å). For the sake of clarity, the same color scheme has been adopted: black for OSW-1, red for U18666A, green for wortmannin, blue for phytol and purple for β-CD. detector (Simbari et al., 2016). Experiments were performed in dupli­ 2.8. SARS-CoV-2 antiviral activity in an in vivo hamster model cate, and each sample was analyzed in triplicate. All animal experiments were approved by the IRTA committee on 2.7. SARS-CoV-2 antiviral activity in a human nasal epithelial model animal experimentation and were authorized by the Generalitat de Cat­ alunya (11964). The researchers adhered to the 3Rs principle and To assess the MβCD antiviral activity in a physiological model, we assured animal welfare throughout the study. The study was evaluated used a human nasal airway epithelium (HNE) model (Mucilair™, Epi­ by the Biosecurity committee of IRTA and was performed under BSL-3 thelix) with the specific medium provided. Inserts were cultured at the conditions. Nine-week-old golden Syrian hamsters (Janvier) were air liquid interface, mimicking in vivo nasal tissue. In this model we divided into 3 groups in a sex-balanced ratio (50 % females): G0 (n = 4; added 2.5 mM MβCD on the apical side or on the basal medium, or 2 µM untreated and uninfected animals), G1 (n = 8; untreated and SARS-CoV- Remdesivir on the basal medium, or left cultures untreated. SARS-CoV-2 2-NanoLuc infected animals), G2 (n = 8; MβCD-treated and SARS-CoV- D614G was added at an MOI= 0.01 to the apical side of each insert for 1 2-NanoLuc infected animals). Animals were inoculated by intranasal h at 37ºC and 5 % CO2. The apical side was then extensively washed with instillation with 50 mM MβCD or PBS (100 μL/individual, 50 μL for each PBS and incubated for 24 h. MβCD on the apical side was washed and not nostril), for the treated and untreated groups, respectively. Then, ani­ replaced thereafter. Drugs added on the basal medium were present mals were inoculated by intranasal instillation with 103 SARS-CoV-2 during the whole experiment and replaced at 48 hpi. Viral content was NanoLuc replicative competent reporter virus (n = 16) resuspended in measured collecting the apical side of inserts after adding 300 μL/well of 50 mM MβCD (for the treated animals) or PBS (for the untreated ani­ PBS at 24-, 48- and 72-hours pi. SARS-CoV-2 nucleocapsid concentration mals; 100 μL/individual, 50 μL for each nostril). The four remaining was measured by ELISA. The cytopathic effect was measured 72 hpi hamsters were intranasally inoculated with PBS (100 μL/individual, 50 using the Cell Titer-Glo Assay to measure ATP released by living cells on μL for each nostril) and used as negative controls. For virological ex­ a DL Ready Luminoskan (ThermoScientific). aminations, 4 hamsters per group (G1-G2) and 2 control hamsters (G0) were euthanized on days 1 and 2 post-infection. Oral swabs were collected from all animals before the challenge and 6 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 Fig. 2. Antiviral activity of compounds tested against HCoV-229E. MRC-5 cells were absorbed with HCoV-229E at MOI of 0.1 PFUs/cell for 1 h, exposed to increasing concentrations of the drug for 24 h and processed by immunofluorescence with antibodies specific for the HCoV-229E N protein and with an anti-rabbit secondary antibody conjugated with Alexa fluor 488 (green). Nuclei were labeled with DAPI (blue). Images were collected with an epifluorescence microscope. (A) Representative pictures of the immunofluorescence assay. Scale bars, 100 µm. (B) Dose-response curves (red lines) of OSW-1, HβCD, U18666A, Phytol, Mdivi-1, FLI 06, Baclogen and CI976 were determined by nonlinear regression. Data is shown as mean ± S.E.M. of 3 biological replicates. Cytotoxic effect on MRC-5 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (black lines). at the euthanasia. At necropsy, samples from nasal turbinate and lung and then 45 cycles of 94 ◦ C for 15 s, 58 ◦ C for 30 s were taken and placed in individual microfuge tubes containing 500 μL Upon euthanasia, animals were necropsied, and lungs, nasal turbi­ of DMEM (GIBCO) supplemented with 1 % penicillin–streptomycin (PS) nate and brain samples were fixed with formalin and routinely stained (GIBCO) and a single zinc-plated, steel, 4.5-mm bead. Samples were with haematoxylin & eosin. Slides were examined under an optical homogenized at 30 Hz for 2 min using a TissueLyser II (QIAGEN GmbH, microscope and analysed in a blinded fashion. Lesions were scored Hilden, Germany) and centrifuged for 30 s at 11,000 rpm. Supernatants following a semi-quantitative approach based on the inflammation were analyzed for Nanoluciferase content with the Nano-Glo Luciferase severity (none, mild, moderate, or severe), as previously described [6, Assay System (Promega) at a 1:1 sample:luciferase ratio and assessed on 56]. a Fluoroskan Ascent FL luminometer (ThermoFisher). Samples were SARS-CoV-2 nucleoprotein (NP) antigen was detected in target tis­ stored at − 70 ◦ C for further analysis. sues with an immunohistochemistry protocol using a rabbit monoclonal Viral RNA was extracted from samples using the IndiMag pathogen antibody (40143-R019, Sino Biological, Beijing, China) at a 1:15,000 kit (Indical Bioscience) on a Biosprint 96 workstation (QIAGEN) ac­ dilution. The amount of NP was scored semi-quantitatively in a blinded cording to the manufacturer’s instructions. RT–PCR used to detect viral fashion (no detection, low amount, moderate amount or high amount of gRNA is based on the one published by Corman et al. Eurosurveillance; antigen [6,56]). 2020;25:2000045, with minor modifications to adapt it to the AgPath-ID Viral titration of replicative SARS-CoV-2-nanoLuc was performed in One- Step RT–PCR Kit (Life Technologies). The primers and probes used, VeroE6 cells. Briefly, homogenized tissue was serially diluted 10-fold and their final concentration are the following: forward: 5′ -ACAGG­ and transferred to an opaque (white) 96-well plate with 70 % conflu­ TACGTTAATAGTTAATAGCGT-3′ [400 nM], reverse: 5′ -ATATTGCAG­ ence of VeroE6 cells and incubated for 48 h at 37 ◦ C and 5 % CO2. Then, CAGTACGCACACA-3′ [400 nM] probe: 5′ -FAM ACACTAGCCATCCTTA samples were incubated for 3 min with Nano-Glo® reagent (Promega) at CTGCGC TTCG-TAMRA-3′ [200 nM]. Thermal cycling was performed at a 1:1 ratio in absence of light. Read-out was performed with a Fluo­ 55 ◦ C for 10 min for reverse transcription, followed by 95 ◦ C for 3 min roskan Ascent FL (Thermo Fisher Scientific). Viral titration of replicative 7 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 Fig. 3. Antiviral activity of drugs against SARS-CoV-2. (A) Cytopathic effect on Vero E6 cells exposed to 200 TCID50/mL of SARS-CoV-2 in the presence of increasing concentrations of OSW-1 (1 – 1,3 ×10-5 µM), HβCD (20 – 0,00026 mM), U18666A (20 – 2,6 ×10-4 µM) and Phytol (100 – 0,78 µM). Non-linear fit to a variable response curve from one representative experiment out of three with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. Cytotoxic effect with the same drug concentrations in the absence of virus is also shown (black lines). The IC50 value is indicated on each graph. IC50 for OSW-1 could not be calculated because 100 % inhibition was not obtained with this compound. (B) Cytopathic effect on Vero E6 cells exposed to 200 TCID50/ mL of different variants of concern of SARS-CoV-2 as described in A, and in presence of Remdesivir. SARS-CoV-2 virus was performed as described elsewhere [19,42] and virus. The designed computational strategy is summarized in Fig. 1, quantified using the Reed-Muench method. including the selected viral targets (Fig. 1A) as well as the chemical This in vivo experiment was also performed using a wildtype SARS- structures of the best-ranked compounds (Fig. 1B). The performed cal­ CoV-2 virus (D614G variant, GISAID ID EPI_ISL-47147). culations suggested that OSW-1 is efficiently anchored at the active site of the Mpro (Fig. 1C). Indeed, that hit exhibited the largest binding en­ 3. Results ergy (ΔG = − 108.79 kcal/mol) among all the small molecules included in our library. Additional large interactions are predicted for the binding 3.1. Molecular modeling for antiviral selection of U18666A, wortmannin and phytol to NPC1 (ΔG in the range of –90 to − 80 kcal/mol). Unfortunately, only moderate interactions are associ­ We first screened the bibliography to identify: (i) molecules targeting ated with other viral targets as Spike, NSP16 and PLPro, with ΔG values RNA viral polymerases or cellular factors used by viruses; (ii) hydro- under the threshold of − 66 kcal/mol. The complete list of the predicted soluble extracts and essential oils from plants used in medicine and binding-free energies for the best-ranked compounds is provided in with antiviral activity; (iii) commercially available drugs that could Tables S2 and S3. have antiviral activity. These studies produced a library of 116 com­ The adopted poses by the best-ranked ligands is illustrated in Fig. 1C pounds that were all further analyzed with molecular models to identify once reached the target. In the case of cyclodextrins, β-CD was selected potential inhibitors of SARS-CoV-2 proteins or cell proteins used by the as a representative macrocycle. The analysis of the produced structures 8 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 Table 2 The energetic analysis offers an unbiased criterium for ligand se­ List of in vitro tested compounds against SARS-CoV-2. lection. However, a large interaction with these targets does not guar­ Drugs IC50 ± SD (µM) First cytotoxic conc. (µM) antee an efficient activity against the virus, given that many other mechanisms may affect the inhibitory ability in the biological scenario Remdesivir 0.51 ± 0.71 100 OSW-1 ~ 0.002 ± 68.41 0.007 [8]. Consequently, selection criteria for testing compounds in cell cul­ Hβ-CD 847.09 ± 2240.10 20000 ture against coronaviruses HCoV-229E and SARS-CoV-2 was completed U18666A ~ 0.17 ± 539.43 20 with additional parameters including their low toxicity and commercial Phytol ~ 140.90 ± 402.29 100–200 availability. With this information, we selected 44 compounds to be Cytotoxic drugs Bortezomib ~ 0.4958 0.8 tested first against HCoV-229E. In addition, we also tested the FLI06 0.0151 4–6.25 hydro-soluble extract and the essential oil from Pinus sylvestris in cells Mdivi-1 ~ 0.8317 20 infected with HCoV-229E, given its described antiviral properties [18, PIK93 ~ 0.005508 20 37]. CI976 0.2997 100 Valinomycin NA 0.32 Dehydrocostus lactone ~ 0.8439 100 3.2. Antiviral activity against HCoV-229E Digitoxin ~ 1.524 1.56 Wortmanin 0.941 25 To test the antiviral activity of selected compounds, MRC-5 cells Terpinolene ~ 2.305 10000 Gingenoside ~ 1.403 50 were infected with HCoV-229E and treated in parallel with increasing No active drugs concentrations of the selected compounds. Drug cytotoxicity was first L-arginine ~ 0.000 NA measured, and safe drug-doses were used to determine the percentage of α-Terpineol ~ 23.67 NA infected cells by immunofluorescence using an antibody specific for the The IC50 median and SD of the viral cytopathic effect from three independent HCoV-229E nucleocapsid protein (Fig. 2A). With these assays, we experiments is provided, and the first drug-cytotoxic concentration is provided calculated the concentration of compound required to inhibit 50 % of for uninfected cells. SD, standard deviation; NA, non-applicable. the virus (IC50), and the concentration for the 50 % cytotoxic effect (CC50) (Fig. 2B). Thirteen compounds, including OSW-1, HP-β-cyclo­ confirms that both OSW-1 and β-CD are in the catalytic pocket of the dextrin (HβCD), U18666A and Phytol inhibited HCoV-229E infection at Mpro, while U18666A, phytol and wortmannin reach the central pocket non-toxic concentrations (Table 1, Fig. 2B top graphs). Mdivi-1, FLI06 of the NPC1. To ensure that ligands remain in their pockets under bio­ and CI976 showed an inhibitory effect but at toxic concentrations, and logical conditions, MD trajectories of 100 nano-seconds (ns) were per­ Baclogen did not have an inhibitory effect on HCoV-229E (Fig. 2B bot­ formed. The stability was monitored through the so-called root means- tom graphs). In the case of BMS309403, Dimercaprol, and alpha-cedrol, square deviation (RMSD) (Fig. 1C, right panel). A close inspection of they inhibited 229E but their CC50 was close to the IC50 and they were RMSD values revealed that both OSW-1 (black line) and βCD (purple discarded for further analysis due to toxicity issues. As indicated in line) are stabilized with a RMSD variation of ca. 2 Å. On the contrary, Table 1, the most potent antiviral was OSW-1 (IC50 of 0.5 nM), followed U18666A, phytol and wortmannin yielded an average RMSD of ca. 4 Å. by U18666A, phytol and HβCD (IC50 of 2.6 µM, 18.6 µM and 4.3 mM, This difference is probably due to the fact that NCP1 has an internal respectively). pocket while the Mpro active site is localized on its surface, which in­ duces a larger conformational change upon ligand binding. However, 3.3. OSW-1, HβCD, U18666A and Phytol show anti- SARS-CoV-2 despite such dissimilarity, all five drugs were quickly equilibrated, with activity in Vero E6 cells a variation of less than 1 Å after the first 20 ns of trajectory, suggesting the drug-target adducts are stable with time, a prerequisite to exhibit Compounds that revealed antiviral activity against HCoV-229E were biological activity. next evaluated against SARS-CoV-2. Vero E6 cells were exposed to Fig. 4. TEM of Vero E6 cells infected with SARS-CoV-2. Ultrathin sections of cells infected with SARS-CoV-2 at MOI of 0.02 PFU/cell and 48 hpi. (A) Overview of a cell with clusters of DMVs (asterisks) near the nucleus (N). (B) High magnification of a group of early DMVs with electron-dense content. (C) Cluster of late DMVs with fibrillar content. (D) Single mem­ brane vesicles with viral particles in their lumen (white arrowheads). (E) Complex vacuole (CV) with viral particles (black arrowheads) in their lumen. The inset shows a higher magnification of one of the viral particles inside the CV. (F) Viral particles (black arrowheads) at the plasma membrane (P). M, mitochondrion, Scale bars, 1 µm in A, 200 in B-F. 9 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 Fig. 5. TEM of Vero E6 cells infected with SARS-CoV-2 and effects of HβCD. Cells infec­ ted with SARS-CoV-2 at MOI of 0.02 PFU/cell were incubated with 0.16 mM (A-D) or 20 mM HβCD (E-I) and prepared for TEM at 48 hpi. (A) Overview of a cell with a cluster of DMVs (asterisk). (B) Group of DMVs with alteration of the inner membrane (arrows) and a single membrane vesicle with a viral particle (white arrowhead) in close vicinity. (C) Complex vac­ uole (CV) with viral particles in the lumen. The inset shows a viral particle at higher magnifi­ cation. (D) Viral particles (black arrowheads) at the plasma membrane (P). (E) Overview of a cell. (F) DMV with a group of single membrane vesicles containing viral particles (white ar­ rowheads) in close vicinity. (G) Group of DMVs with alteration of the inner membrane (arrow). (H) Complex vacuole with viral particles (ar­ rowheads) in the lumen. (I) Viral particles (black arrowheads) at the plasma membrane (P). N, nucleus. Scale bars, 1 µm in A and E; 200 nm in B-D, F-I. SARS-CoV-2 in the presence of increasing concentrations of the different swelling of Golgi cisternae and lysosomes were observed when the drugs. After three days, the cytopathic effect of the virus and the cyto­ higher dose is applied (Fig. S6D-F). For phytol analysis, infected cells toxic effect of the drugs on cells were analyzed and the IC50 value were incubated also with two different concentrations. Cells treated calculated for each drug at non-cytotoxic concentrations. Results with the highest concentration showed clear signs of cytopathic effect showed that OSW-1, HβCD, U18666A and Phytol had antiviral activity and only the cells treated with the lowest concentration were processed on these cells (Fig. 3A), while the rest of tested compounds were not for TEM. In phytol-treated cells, DMVs were altered to some extent but active or were cytotoxic (Fig. S1 and Table 2). The antiviral activity of treatment had minor effects in virus assembly and egress (Fig. S7A-E and these four drugs was similar when tested for different variants of Table S4). In mock-infected cells, phytol did not cause significant al­ concern of SARS-CoV-2 and comparable to the control Remdesivir terations, with the only exception of mild increase in the amount of (Fig. 3B). The antiviral activity found for tested compounds with both cytosolic glycogen granules and lipid droplets (Fig. S7F-I). HCoV-229E and SARS-CoV-2 is summarized in Tables 1 and 2. When SARS-CoV-2 exposed cells were treated with HβCD at the Then we investigated the mechanism of action of the four selected suboptimal concentration of 0.16 mM, DMVs showed alterations of the antivirals active against α and β coronavirus. We first performed inner membrane (Fig. 5A and B). Viruses inside SMVs appear normal, morphological studies of Vero E6 cells infected with SARS-CoV-2 by but large groups of distorted viral particles were seen inside complex transmission electron microscopy (TEM). These studies showed char­ vacuoles (Fig. 5C). Extracellular virions attached to the plasma mem­ acteristic structures assembled by SARS-CoV-2, which are: double- brane looked normal (Fig. 5D). At inhibiting concentrations using membrane vesicles (DMVs) where the virus replicates its genome 20 mM of HβCD, all viral structures were reduced (Fig. 5E and Table S4) (Fig. 4A-C), viral particles inside single-membrane vesicles (SMVs) and a few detected DMVs showed membrane alterations (Fig. 5F and G). (Fig. 4D), complex vacuoles (CV) with viruses (Fig. 4E) and extracellular Intracellular viral particles inside SMVs (Fig. 5F), CVs (Fig. 5H) and viral particles (VPs) (Fig. 4F). extracellular virions (Fig. 5I) were seen in low numbers compared with TEM of SARS-CoV-2 infected cells treated with two different con­ infected non-treated cells (Table S4). These results show that HβCD af­ centrations of OSW-1 (Fig. S2 and Table S4) and of U18666A (Fig. S3 fects the biogenesis of DMVs and the subsequent morphogenesis of new and Table S4) suggested that in Vero E6 cells, these compounds block viral particles, an observation that could be linked to the capacity of this DMVs’ assembly and function, which would affect subsequent steps of compound to reduce viral fusion and alter cellular membranes. At the viral morphogenesis. Mock-infected cells incubated with OSW-1 lowest concentration, HβCD did not produce appreciable changes in cell exhibited normal morphology and no alterations were detected in any compartments of mock-infected cells (Fig. S8A-C). However, non- of the cell organelles compared to mock-infected cells in the absence of infected cells with 20 mM of HβCD had increased number of vacuoles the drug (Fig. S4 and S5). Incubation with the lower dose of U18666A (Fig. S8D), swelling of ER, Golgi and lysosomes (Fig. S8E and F). caused no appreciable alterations in mock-infected cells (Fig. S6A-C) but 10 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 Fig. 6. Drug inhibition of pseudovirus entry in ACE2–293 T cells and of SARS-CoV-2 infection in pulmonary cells. (A) Relative viral entry of SARS-CoV-2 pseudoviruses in the presence of the indicated drugs in ACE2 expressing HEK-293 T cells. Cells were exposed to fixed amounts of SARS-CoV-2 Spike lentiviruses in the presence of decreasing drug concentrations. Values show luciferase expression of the reporter lentiviruses pseudotyped with SARS-CoV-2, normalized to the luciferase expression of mock-treated cells (set at 100 %). Mean and standard deviation from two experiments with two replicates each are represented, excluding cytotoxic values. (B) Relative viral replication of SARS-CoV-2 assessed on CaLu-3 cells in the presence of the indicated drugs. After 24 h of adding virus and drugs at the indicated concentrations, cells were washed and compounds were added at the same final concentration for an additional 48 h. Then supernatants were tested for viral release by detecting SARS-CoV-2 nucleocapsid concentration by ELISA. Values are normalized to the nucleocapsid concentration by mock-treated cells (set at 100 %), which reached 5716 ± 2237 pg/mL (mean ± SD). Mean and standard deviation from four experiments are represented, excluding cytotoxic values. 3.4. HβCD and U18666A block viral fusion, but only HβCD inhibits 3.5. Other members of the Cyclodextrin family also inhibit SARS-CoV-2 infection in pulmonary cells infection in pulmonary cells We therefore assessed the capability of these four drugs to inhibit the Given the well-known safety profiles of different types of β-cyclo­ entry of SARS-CoV-2 pseudovirus in ACE2 expressing HEK-293T cells. dextrins, that have been used as excipients in the pharmaceutical in­ Cells were exposed to fixed amounts of lentiviruses pseudotyped with dustry for decades [26], we next aimed to test whether other SARS-CoV-2 spike in the presence of decreasing drug concentrations. cyclodextrins could hold the potential to inhibit viral replication. To test Results showed that at drug concentrations without any cytotoxic effect, if other cyclodextrins could inhibit SARS-Cov-2 pseudoviral entry, we HβCD and U18666A inhibited the entry of pseudovirus, while OSW-1 performed studies in ACE2 expressing HEK-293 T cells. At no cytotoxic and Phytol did not (Fig. 6A). Cytopathic effect of the drugs in the concentrations (Fig. S9C), seven cyclodextrins inhibited the pseudoviral absence of virus is shown Fig. S9A. These findings with the functional SARS-CoV-2 entry, being the most potent β, HP-β, HP-β and meth­ pseudoviral assay suggest that the mechanism of action of cyclodextrins yl-β-CDs (Fig. 7A). is to reduce viral fusion. Next, we assayed if the most active cyclodextrins inhibiting pseu­ We next tested SARS-CoV-2 antiviral activity in pulmonary Calu-3 doviral fusion could also block SARS-CoV-2 replication in pulmonary cells as a more physiological and relevant cellular model. After incu­ Calu3 cells. The quantification of SARS-CoV-2 nucleoprotein released to bating these cells with the drugs and the virus for 24 h, the virus was the supernatant detected by ELISA revealed that β-CDs, HP-β-CDs, and washed away and compounds were added at the same concentration for methyl-β-CDs inhibited the SARS-CoV-2 viral activity on Calu-3 cells, for 48 h. The amount of SARS-CoV-2 nucleoprotein released to the super­ the D614G (Fig. 7B) and the Omicron BA.1 variant of concern (Fig. 7C) natant was then measured by ELISA (Fig. 6B) and the cytopathic effect of at no cytotoxic concentrations. These results further highlight the po­ the drugs in the absence of virus in Calu-3 cells was assessed by lumi­ tential of the β cyclodextrin family as antivirals against SARS-CoV-2. nescence (Fig. S9B). Only HP-β-CD at 10 mM effectively inhibited SARS- CoV-2 viral release into the supernatant of Calu-3 cells as observed for 3.6. Cyclodextrins inhibit SARS-CoV-2 replication by interfering with control remdesivir, while no inhibition was observed for the other viral fusion via cholesterol depletion compounds (Fig. S9D) at non-cytotoxic concentrations. Hence, HP-β-CD resulted as the most promising candidate to block SARS-CoV-2 replica­ We had previously found that β-CD are in the catalytic pocket of the tion on pulmonary cells. Mpro of SARS-CoV-2 by molecular modeling (Fig. 1C). This finding pointed to a possible explanation for the antiviral activity detected. All cyclodextrins, including α, β, β, HP- α, HP-β, HP-β and methyl-β-CDs, 11 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 Fig. 7. Members of the Cyclodextrin family inhibit SARS-CoV-2 infection in pulmonary cells. (A) Relative viral entry of SARS-CoV-2 pseudoviruses in the presence of the indicated cyclodextrins in ACE2 expressing HEK-293 T cells. Cells were exposed to fixed amounts of SARS-CoV-2 Spike lentiviruses in the presence of decreasing drug concentrations. Values show luciferase expression of the reporter lentiviruses pseudotyped with SARS-CoV-2, normalized to the luciferase expressi % on of mock-treated cells (set at 100 %). Mean and standard deviation from two experiments with two replicates each are represented, excluding cytotoxic values. (B) Relative viral replication of D614G (C) or Omicron (D) SARS-CoV-2 variant was assessed on CaLu-3 cells in the presence of the indicated cyclodextrins. After 24 h of adding virus and drugs at the indicated concentrations, cells were washed and compounds were added at the same final concentration for additional 48 h. Then supernatants were tested for viral release by detecting SARS-CoV-2 nucleocapsid concentration by ELISA. Values are normalized to the nucleocapsid concentration by mock-treated cells (set at 100 %), which reached 5716 ± 2237 pg/mL (mean ± SD). Mean and standard deviation from three experiments are represented, excluding cytotoxic values. 12 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 Fig. 8. Cyclodextrins inhibit SARS-CoV-2 replication by interfering with viral fusion via cholesterol depletion. (A) Mpro activity measured in presence of increasing concentrations of methyl-β-CD (MβCD) or GC376 as positive inhibitor. Results are represented as the percentage of inhibition of Mpro activity in the absence of drugs. (B) Lipidomic measurement of plasma membranes from Calu3 cells treated or not with MβCD for 2 h at 37ºC and 5 % CO2. were screened by molecular modeling against the SARS-CoV-2 Mpro. incubated for 72 h. Remdesivir was added to the basal medium, while Since it is possible to inhibit the action of Mpro by targeting two allosteric other inserts were left untreated. Viral replication was monitored at 48 sites -PDB codes 7AXM and 7AGA- [17]), we expanded the chemical and 72 hpi by measuring SARS-CoV-2 nucleocapsid concentration by space of search into the designed models. Theory predicts that cyclo­ ELISA. SARS-CoV-2 released to the apical supernatant picked at 48 hpi dextrins can bind both active and allosteric Mpro sites, although signif­ and decreased at 72 hpi (Fig. 9B-C). Initial MβCD addition to the apical icant dissimilarities appear depending on the size and nature of the side reduced viral replication by 74 % at 48 h pi (Fig. 9B), while con­ macrocycle (Table S3). To test whether this interaction occurred in vitro, stant presence of MβCD in the basal side almost had no effect (11 %) as the Mpro activity was measured in presence of increasing concentrations opposed to the condition with remdesivir that completely blocked it of methyl-β-CD (MβCD). While MβCD did not show activity, active (Fig. 9B-C). The cytopathic effect measured at 72 hpi indicated the HNE GC376 control inhibited Mpro in a dose dependent manner (Fig. 8A). cells were alive with all the tested conditions (Fig. S9E). These results We next explored alternative antiviral mechanisms of action of cy­ further emphasize the potential use of MβCD as a prophylactic antiviral clodextrins. Given the well-known capacity of cyclodextrins to extract in nasal sprays against SARS-CoV-2. cholesterol from biological membranes [28], we performed a lipidomic analysis focusing on different lipids associated to cholesterol enriched 3.8. Prophylaxis treatment with Methyl-β-cyclodextrin protects animals domains in biological membranes. Calu-3 cells treated with increasing from SARS-CoV-2 infection concentrations of MβCD showed a reduction of free and ester cholesterol along with sphingomyelin, and did not affect phosphatidylcholine To test the capacity of MβCD to prevent SARS-CoV-2 infection in vivo, (Fig. 8B). These results further confirm the capacity of cyclodextrins to we tested its prophylactic activity in hamsters. Animals were pre-treated alter the composition of cholesterol enriched domains actively involved with MβCD or PBS intra-nasally before challenging via the same route in viral fusion processes. Taken together, these experiments along the with a SARS-CoV-2 reporter virus that produces nano-luciferase upon pseudoviral fusion assays highlight the potential of cyclodextrins to infection. Animals were euthanized on day 1 and 2 post infection and inhibit SARS-CoV-2 replication by interfering with viral fusion via compared to uninfected controls, measuring the amount of nano­ cholesterol depletion. luciferase content in nasal turbinates and lungs (Fig. 10A-B). On day 1, the four infected untreated animals had detectable nanoluciferase levels 3.7. Methyl-β-cyclodextrin inhibits SARS-CoV-2 replication in a human both at the nasal turbinates and lungs, while no nanoluciferase signal nasal epithelial model was detected on MβCD treated animals. Similar results were obtained on day 2 post-infection, where higher levels of nanoluciferase were detec­ We aimed to test the antiviral effect of MβCD in a more physiologi­ ted in the nasal turbinates and lungs of untreated infected animals, and cally relevant model. We used a human nasal epithelial (HNE) model no sign of infection was detected on MβCD treated animals (Fig. 10A-B). which mimics the characteristics of in vivo nasal tissue. In this HNE These results were further confirmed by qPCR on days 1 and 2 post- model we added MβCD, either on the apical side or the basal medium of infection in nasal turbinates and lungs (Fig. 10C-D), and by viral the culture (Fig. 9A), just before adding SARS-CoV-2 to the apical side infectivity (Fig. 10E-F). Similar protection trend was observed in the for 1 h. Apical side was then extensively washed and inserts were histopathology and haematoxylin and eosin-stained sections of nasal 13 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 Fig. 9. Methyl-β-cyclodextrin inhibit SARS-CoV-2 replication in a human nasal epithelial (HNE) model. (A) Schematic representation of the HNE model used, showing the apical side, the basal medium and the cells cultured in the air-liquid interphase. (B) SARS-CoV-2 replication in the HNE model in the presence of 2.5 mM MβCD either on the apical side (red) or on the basal medium (orange), or 2.5 uM Remdesivir (green) on the basal medium, without drugs (blue), or in the absence of virus (black). SARS-CoV-2 was added to the apical side for 1 h, extensively washed afterwards, and nucleocapsid concentration was measured by ELISA at 24, 48 and 72 hpi. (C) The results of two independent experiments following the same procedure explained in ‘B’ and measured at 72 hpi. turbinates and lungs of MβCD-treated animals as compared to controls different protocols of infection and drug treatment can be tested, which (Fig. 10G-J). Protective trend was also observed with a replicative SARS- increases the probabilities of identifying molecules with antiviral ac­ CoV-2 isolate specially in the lung tissue (Fig. S10), but with a less tivity. We have used a workflow involving biocomputational analysis potent prophylactic effect as compared to the Nanoluciferase virus. and several biological assays to carefully select potential antivirals These results further confirm the prophylactic capacity of MβCD to against different coronaviruses, including HCoV-229E and SARS-CoV-2. protect from SARS-CoV-2 infection in the nasal epithelium of hamsters. From our list of 116 compounds that target cell factors and pathways, 4 showed antiviral activity against both coronaviruses. Results with β-CDs 4. Discussion were particularly relevant as showed consistent efficacy in different cellular models including human pulmonary cells and the nasal Although pathogenic viruses pose a real and growing threat to public epithelium of hamsters in vivo. Particularly promising is our finding health, we have few medicines to prevent and treat viral infections. suggesting that the mechanism of action of β-CDs is interfering with viral Here, a library of potential inhibitors of coronavirus infection was fusion. Results from lipidomic analysis and transmission electron mi­ elaborated with a “based-on-knowledge” strategy. Examining the croscopy (TEM) showed that β-CDs may interfere with coronavirus available information about what viruses use to complete their life cycle infection by altering cholesterol and sphingomyelin content and dis­ in cells and the description of the mechanism of action of drugs, we rupting the organization of membranes used by the virus. This is sup­ found compounds with potential to be used as antivirals to treat coro­ ported by our TEM results that showed dose-dependent effects of β-CDs navirus infections. The main advantage of this strategy compared to in all SARS-CoV-2 structures in infected cells. The integrity of DMVs was high-throughput analysis is that the list of candidates is limited, and that compromised and viral morphogenesis was impaired, with the 14 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 Fig. 10. Methyl-β-cyclodextrin nasal application inhibits SARS-CoV-2 replication in a hamster model. Hamsters were intranasally adminis­ tered with or without MβCD at 50 mM and challenged with a SARS-CoV-2 Nanoluciferase reporter virus. Control uninfected animals were also assayed. (A-B) At 1- or 2-days post-infection, nasal turbinates and lungs collected from euthanized animals were lysed with the Nano-glo Luciferase system (Promega) and luminescence was measured with a plate reader in rela­ tive light units (RLUs). (C-J) At 1- or 2- days post-infection, nasal turbinates and lungs collected from euthanized animals were analysed for (C-D) viral RNA presence in inverted CTs by qPCR, (E-F) by viral infectivity, (G-H) by immunohistochemistry to detect the NP of SARS-CoV-2 and (I-J) by histopathological observation in hae­ matoxylin and eosin-stained sections. 15 D. Raïch-Regué et al. Biomedicine & Pharmacotherapy 164 (2023) 114997 production of abnormal viral particles that are sometimes trapped in Investigation. Cristina Lorca-Oró: Methodology, Investigation. Júlia DMVs and inside large vacuoles that could represent degradation com­ Vergara-Alert: Conceptualization. Joaquim Segalés: Conceptualiza­ partments. Our results confirm and broaden recent findings focused on tion. Jorge Carrillo: Conceptualization. Julià Blanco: Conceptualiza­ HP-β-CDs [4], and with different methods further expand these results tion. Bonaventura Clotet Sala: Conceptualization, Resources, Funding encompassing other members of the cyclodextrin family and SARS-CoV2 acquisition. José P. Cerón-Carrasco: Conceptualization, Investigation, VOCs, including Omicron. Formal analysis, Funding acquisition, Writing – review & editing. Nuria Given that cyclodextrins are suited for oral, nasal or nebulized so­ Izquierdo-Useros: Conceptualization, Supervision, Funding acquisi­ lutions, these results open avenues to test diverse drug formulations tion, Writing – review & editing. Cristina Risco: Conceptualization, known to be safe in humans [50,53]. The reduced infection after MβCD Supervision, Funding acquisition, Writing – review & editing. prophylactic addition on a human nasal airway epithelium and in a physiologically relevant hamster model shown herein further supports Declaration of Competing Interest the clinical testing of these compounds. To the best of our knowledge this is the first set of data that shows the efficacy of MβCD in inhibiting The authors declare the following financial interests/personal re­ viral infection in the nasal epithelium and upper respiratory tract of an lationships which may be considered as potential competing interests: animal model. The well-known safety profiles of β-CDs render these CR, IFC, MS, RT, PO-G, AF-O, SF-S, NI-U, DP-Z, JM-B, DR-R, JPC-C, JAG molecules as ideal candidates to develop affordable prophylactic com­ and EN-D are inventors of a patent application for use of cyclodextrins to pounds against coronaviruses [11,49]. Such drugs, which are already treat viral infection (PCT/EP2023/057735). The authors declare no approved for clinical use in nasal spray devices [16,36], may be easier to other related competing interest. deploy in low income countries compared to vaccines, which often require cold storage and must be administered by trained personnel. Acknowledgements Given that β-CDs are widely used for compound encapsulation, they could be easily combined with other antivirals to potentiate activity and Special thanks to Dr. Mª Carmen Martínez Jiménez, from the Hospital avoid viral resistance. Finally, the broad-spectrum mechanism of action Nuestra Señora de Sonsoles de Avila for sharing knowledge about the shown herein, which inhibits viral fusion with cellular membranes, use of plant extracts and their active compounds to treat viral infections. could help to counteract other respiratory viruses, providing an arsenal We are grateful to Jorge Díaz for his constant support at the CMCIB to deploy in front of new variants of concern or future novel coronavi­ facility. We thank the animal facility technicians A. Pou and Ò. García, ruses with pandemic potential. Broad-spectrum antivirals such as β-CDs the pathological anatomy technicians A. Barceló and R. López and the could be ultimately applied to counteract unknown emergent viruses yet laboratory management unit I. Cordón, M. Mora an X. Abad for their to appear, but will need rigorous clinical assessment for further support with the in vivo experiments at IRTA-CReSA. We thank Rytis development. Boreika for editing the manuscript. Funding Appendix A. Supporting information This work has been funded by grant RTI2018-094445-B100 (MCIU/ Supplementary data associated with this article can be found in the AEI/FEDER, UE) from the Ministry of Science and Innovation of Spain online version at doi:10.1016/j.biopha.2023.114997. (C.R.), by Palex Medical S.A., Sika S.A.U. and 7 more companies, and by Ms. Raquel Casaus Alvarez, Mr. Miguel Pardo Gil, Mr. Jacques Noguès References and a total of 2916 citizens through the Precipita crowdfunding platform of Fecyt (Fundación Española para la Ciencia y la Tecnología). NI-U is [1] A. Agarwal, B. Rochwerg, F. Lamontagne, R.A.C. Siemieniuk, T. Agoritsas, L. Askie, supported by the Spanish Ministry of Science and Innovation (grant L. Lytvyn, Y.-S. Leo, H. Macdonald, L. Zeng, W. Amin, F.A.J. Barragan, F.J. Bausch, PID2020-117145RB-I00), EU HORIZON-HLTH-2021-CORONA-01 E. Burhan, C.S. Calfee, M. Cecconi, D. Chanda, V.Q. Dat, A. De Sutter, B. Du, H. Geduld, P. Gee, N. Harley, M. Hashmi, B. Hunt, F. Jehan, S.K. Kabra, S. Kanda, (grant 101046118) and by institutional funding of Grifols, Pharma Mar, Y.-J. Kim, N. Kissoon, S. Krishna, K. Kuppalli, A. Kwizera, T. Lisboa, I. Mahaka, HIPRA, Amassence and Palobiofarma. This work used the computational H. Manai, G. Mino, E. Nsutebu, J. Preller, N. Pshenichnaya, N. Qadir, S. Sabzwari, resources of the Centro de Supercomputació n de Galicia (CESGA) sup­ R. Sarin, M. Shankar-Hari, M. Sharland, Y. Shen, S.S. Ranganathan, J.P. Souza, ported by the Partnership for Advanced Computing in Europe (PRACE) M. Stegemann, R. Swanstrom, S. Ugarte, S. Venkatapuram, D. Vuyiseka, A. Wijewickrama, B. Maguire, D. Zeraatkar, J.J. Bartoszko, L. Ge, R. Brignardello- COVID-19 Fast Track Call for Proposals – Allocation Decision – Proposal Petersen, A. Owen, G. Guyatt, J. Diaz, L. Kawano-Dourado, M. Jacobs, P. COVID19-85. O. 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