Identification of SARS-Cov-2 Receptor Binding Inhibitors by in Vitro Screening of Drug Libraries

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Article Identiﬁcation of SARS-CoV-2 Receptor Binding Inhibitors by In Vitro Screening of Drug Libraries

Alon Ben David 1,†, Eran Diamant 1,†, Eyal Dor 1, Ada Barnea 1, Niva Natan 1, Lilach Levin 1, Shira Chapman 2, Lilach Cherry Mimran 1, Eyal Epstein 1, Ran Zichel 1,* and Amram Torgeman 1,*

1 Department of Biotechnology, Israel Institute for Biological Chemical and Environmental Sciences, Ness Ziona 7410001, Israel; [email protected] (A.B.D.); [email protected] (E.D.); [email protected] (E.D.); [email protected] (A.B.); [email protected] (N.N.); [email protected] (L.L.); [email protected] (L.C.M.); [email protected] (E.E.) 2 Department of Pharmacology, Israel Institute for Biological, Chemical and Environmental Sciences, Ness Ziona 7410001, Israel; [email protected] * Correspondence: [email protected] (R.Z.); [email protected] (A.T.); Tel.: +972-8-938-1515 (A.T.) † These authors contributed equally.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the Citation: David, A.B.; Diamant, E.; high-throughput screening of two compound libraries (LOPAC®1280 and DiscoveryProbeTM). Three Dor, E.; Barnea, A.; Natan, N.; Levin, compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an L.; Chapman, S.; Mimran, L.C.; effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction Epstein, E.; Zichel, R.; et al. assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy Identification of SARS-CoV-2 of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these Receptor Binding Inhibitors by In Vitro Screening of Drug Libraries. compounds in the RBD. In light of these findings, the screening system described herein can be Molecules 2021, 26, 3213. https:// applied to other drug libraries to discover potent SARS-CoV-2 inhibitors. doi.org/10.3390/molecules26113213 Keywords: SARS-CoV-2; COVID-19; drug repurposing; spike protein; receptor binding domain Academic Editor: Jan Brezovsky (RBD); angiotensin-converting enzyme 2 (ACE2), high-throughput screening; small molecule inhibitors (SMIs) Received: 2 May 2021 Accepted: 24 May 2021 Published: 27 May 2021 1. Introduction Publisher’s Note: MDPI stays neutral Coronavirus disease 2019 (COVID-19) is a worldwide pandemic caused by severe with regard to jurisdictional claims in acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, SARS-CoV-2 has published maps and institutional affil- infected over 150 million people, and over 3 million deaths have been reported [1]. The iations. emergence of COVID-19 has prompted a global effort to develop vaccines and drugs for prevention and treatment. These efforts have led to the development of vaccines, several of which are being administered worldwide under emergency use authorization [2–4]. How- ever, only a few therapeutics (Remdesivir and monoclonal antibodies) are available under Copyright: © 2021 by the authors. emergency-use or conditional marketing authorization by the American and European Licensee MDPI, Basel, Switzerland. regulatory authorities [5–7]. This article is an open access article SARS-CoV-2 infects host cells via the binding of its spike glycoprotein (S) to human distributed under the terms and angiotensin-converting enzyme 2 (ACE2) on target cell surfaces [8,9]. The spike protein is conditions of the Creative Commons composed of two functional subunits, S1 and S2. S1 includes the ACE2 binding domain Attribution (CC BY) license (https:// (RBD), and the S2 subunit mediates the fusion of the virus to the infected cell membrane [9]. creativecommons.org/licenses/by/ 4.0/). Due to its pivotal role in the virus infection process, the spike and, specifically, the RBD are

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major targets for generating vaccines and antibodies aiming to block the ACE2–spike interaction [2–4]. However, drug development, optimization, manufacturing, and preclinical and clinical studies are time consuming [10]. Alternatively, repurposing approved drugs for treating COVID-19 can potentially promote rapid and safe therapy [11,12]. An efficient strategy for drug repurposing is the high-throughput screening of drug libraries. Recent library screening studies have reported the discovery of several SARS- CoV-2 replication inhibitors [13] and compounds that target host proteins and exhibit antiviral activity by interfering with their interaction with viral proteins [14]. However, no compounds that can inhibit the RBD-ACE2 interaction have been found in these reports. Potential RBD-ACE2 inhibitors have been predicted in several in silico library screening studies only [15–17]. The addition of an in vitro screen of drug libraries can promote the discovery of potent inhibitors of the RBD-ACE2 interaction by gaining a functional layer of data. The urgent need for a treatment prompted us to screen for approved drugs and well- known pharmaceutically active compounds to discover potential inhibitors of SARS-CoV-2 cell attachment in this study. To this end, an in vitro RBD-ACE2 binding assay was established and used to discover inhibitors of the interaction via the high-throughput screening of two compound libraries. One library, LOPAC®1280, contains 1280 pharmaceutically active compounds, and the other, DiscoveryProbeTM, contains 1363 FDA-approved drugs. We report herein the identification of three compounds (heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid) that exhibit high inhibition activities. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. A possible mechanism via molecular docking to the RBD is described.

2. Results 2.1. Experimental Design The identification of compounds that inhibit the binding of RBD to ACE2 was carried out by high-throughput screening of the LOPAC®1280 and DiscoveryProbeTM libraries using an assay designed to specifically measure the RBD-ACE2 interaction. This assay consisted of the SARS-CoV-2 receptor ACE2 adsorbed to 96-well plates and soluble hFc- RBD (Figure1). The first step of the assay included preincubation of hFc-RBD with the tested compounds. The second step included incubation of the mixtures with the adsorbed ACE2. Finally, following a washing step, the RBD-ACE2 interaction was quantified by the addition of a detecting alkaline phosphatase-conjugated goat anti-human-Fc antibody. In the presence of a compound that could interfere with the RBD-ACE2 interaction, a lower signal was obtained. The hit compounds were further verified for their inhibitory effect by

Molecules 2021, 26, x FOR PEER REVIEWa plaque reduction assay conducted in Vero E6 cells with a surrogate3 virus of 12 (rVSV-SARS- CoV-2-S) expressing the SAR-CoV-2 spike protein.

Figure 1. Schematic illustration of the RBD-ACE2 binding assay. Ninety-six-well plates were coatedFigure with 1. ACE2Schematic (100 ng/well) illustration and blocked of the with RBD-ACE2 TSTA buffer. binding Each compound assay. Ninety-six-well (10 µM) from the plates were coated librarieswithACE2 was incubated (100 ng/well) with Fc-RBD and blocked(1 µg/mL) withfor 1 h TSTA at 25 °C. buffer. Following Each incubation, compound mixtures (10 µM) from the libraries were added to the ACE2-coated plates and incubated for 1 h at 37 °C. After washing, the plates were incubated for 1 h at 37 °C with the alkaline phosphatase-conjugated goat anti-human Fc fragment. The plates were then washed, and a colorimetric reaction was developed by the addition of pNPP (measured at 405 nm). Left panel: a maximum signal of an uninterrupted RBD-ACE2 interaction was obtained in the presence of noninhibitory compounds. Right panel: a reduced signal of an inhibited RBD-ACE2 interaction was obtained in the presence of inhibitory compounds.

2.2. Screening the LOPAC®1280 and DiscoveryProbeTM Libraries for RBD-ACE2 Binding Inhibitors The developed binding assay was used to screen two compound libraries for inhibitors of the RBD-ACE2 interaction. The relative binding in the presence of the tested compound (10 µM) was calculated by dividing the absorbance of the compound-containing well by that of the no-compound control wells (Figure 2A). The z-prime parameter of the screened plates, indicating the quality and power of the screening assay, was in the high acceptable range values (0.65–0.97) (Figure 2B).

Figure 2. High-throughput screening of the LOPAC® 1280 and DiscoveryProbeTM compound libraries for inhibitors of the RBD-ACE2 interaction. Each compound was tested at 10 µM. (A) Distribu- tion of the relative RBD-ACE2 binding values in the presence of the compounds. (B) Distribution of the Z-prime values of each plate. The purple and blue dots correspond to LOPAC® 1280 and DiscoveryProbeTM, respectively.

Applying an initial threshold of >40% inhibition (relative binding lower than 60%, Figure 2A) resulted in the selection of 30 and 22 compounds from the LOPAC® 1280 and DiscoveryProbeTM libraries, respectively. These hits were reassayed, and three molecules—heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid—exhibiting a binding inhibition above 60% were further analyzed at different concentrations to determine their 50% inhibitory concentration (IC50) values. The three compounds displayed

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Figure 1. Schematic illustration of the RBD-ACE2 binding assay. Ninety-six-well plates were coated with ACE2 (100 ng/well) and blocked with TSTA buffer. Each compound (10 µM) from the waslibraries incubated was incubated with Fc-RBD with (1 µ Fcg/mL)-RBD for (1 1 µg/mL) h at 25 ◦ C.for Following 1 h at 25 incubation,°C. Following mixtures incubation, were added mixtures towere the added ACE2-coated to the platesACE2 and-coated incubated plates for and 1 h incubated at 37 ◦C. After for 1 washing, h at 37 °C. the platesAfter washing, were incubated the plates forwere 1 h incubated at 37 ◦C with for the1 h alkaline at 37 °C phosphatase-conjugated with the alkaline phosphatase goat anti-human-conjugated Fc fragment. goat anti The-human plates Fc frag- werement. then The washed, plates were and a t colorimetrichen washed, reaction and a was colorimetric developed reaction by the addition was developed of pNPP (measured by the addition at of 405pNPP nm). (measured Left panel: at a 405 maximum nm). Left signal panel: of an a uninterrupted maximum signal RBD-ACE2 of an interactionuninterrupted was obtainedRBD-ACE2 in inter- theaction presence was obtained of noninhibitory in the presence compounds. of noninhibitory Right panel: a reduced compounds. signal ofRight an inhibited panel: a RBD-ACE2 reduced signal of interactionan inhibited was RBD obtained-ACE2 in interaction the presence was of inhibitory obtained compounds. in the presence of inhibitory compounds.

2.2. Screening the LOPAC®1280 and DiscoveryProbeTM Libraries for RBD-ACE2 Binding 2.2. Screening the LOPAC®1280 and DiscoveryProbeTM Libraries for RBD-ACE2 BindingInhibitors Inhibitors TheThe developed developed binding binding assay assay was usedwas toused screen to screen two compound two compound libraries for libraries inhibitors for inhibi- oftors the of RBD-ACE2 the RBD-ACE2 interaction. interaction. The relative The binding relative in binding the presence in the of presence the tested compoundof the tested com- (10poundµM) (10 was µM) calculated was calculated by dividing by the dividing absorbance the ofabsorbance the compound-containing of the compound well-containing by thatwell of by the that no-compound of the no-compound control wells control (Figure wells2A). The (Figure z-prime 2A). parameter The z-prime of the screenedparameter of the plates,screened indicating plates, theindicating quality and the power quality of and the screening power of assay, the screening was in the highassay, acceptable was in the high rangeacceptable values range (0.65–0.97) values (Figure (0.652–B).0.97) (Figure 2B).

Figure 2. 2.High-throughput High-throughput screening screening of the of LOPAC the LOPAC®1280 and® 1280 DiscoveryProbe and DiscoveryProbeTM compoundTM compound libraries librar- fories inhibitorsfor inhibitors of the of RBD-ACE2 the RBD-ACE2 interaction. interaction. Each compound Each compound was tested was at 10testedµM. (atA )10 Distribution µM. (A) Distribu- oftion the ofrelative the relative RBD-ACE2 RBD-ACE2 binding binding values values in the in presence the presence of the compounds.of the compounds. (B) Distribution (B) Distribution of the Z-primeZ-prime valuesvalues of of each each plate. plate. The The purple purple and and blue blue dots dots correspond correspond to LOPAC to LOPAC®1280® 1280 and and DiscoveryProbeTMTM, respectively., respectively.

ApplyingApplying an an initial initial threshold threshold of >40%of >40% inhibition inhibition (relative (relative binding binding lower thanlower 60%, than 60%, ® Figure2 A)2A) resulted resulted in thein the selection selection of 30 of and 30 22and compounds 22 compounds from the from LOPAC the LOPAC1280 and® 1280 and DiscoveryProbeTM libraries, respectively. These hits were reassayed, and three molecules— DiscoveryProbeTM libraries, respectively. These hits were reassayed, and three mole- heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid—exhibiting a binding inhibitioncules—heparin above sodium,60% were aurintricarboxylic further analyzed at acid different (ATA) concentrations, and ellagic to acid determine—exhibiting a binding inhibition above 60% were further analyzed at different concentrations to deter- their 50% inhibitory concentration (IC50) values. The three compounds displayed typ- icalmine dose–response their 50% inh curvesibitory (Figure concentration3 and Table1 ).(IC ATA50) values. yielded theThe most three potent compounds IC 50 value displayed (0.6 µg/mL), followed by ellagic acid (2.5 µg/mL) and heparin sodium (5.5 µg/mL). The maximal binding inhibition of ATA and ellagic acid was ~80%, whereas heparin sodium exhibited a 63% inhibition.

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typical dose–response curves (Figure 3 and Table 1). ATA yielded the most potent IC50 typical dose–response curves (Figure 3 and Table 1). ATA yielded the most potent IC50 Molecules 2021, 26, 3213 typicaltypicalvalue dose–responsedose–response (0.6 µg/mL) curvescurves, followed (Figure(Figure by ellagic 33 andand TableacidTable (2.5 1).1). µg ATAATA/mL) yieldedyielded and heparin thethe mostmost sodium potentpotent (5.5 ICIC µg5050 4/mL) of 12. value (0.6 µg/mL), followed by ellagic acid (2.5 µg/mL) and heparin sodium (5.5 µg/mL). valuevalue (0.6The(0.6 µg/mL),maximalµg/mL), followedfollowed binding byinhibitionby ellagicellagic acidacidof ATA (2.5(2.5 and µg/mL)µg/mL) ellagic andand acid heparinheparin was ~80%, sodiumsodium whereas (5.5(5.5 µg/mL).µg/mL). heparin so- The maximal binding inhibition of ATA and ellagic acid was ~80%, whereas heparin so- TheThe maximalmaximaldium exhibited bindingbinding ainhibitioninhibition 63% inhibition. ofof ATAATA andand ellagicellagic acidacid waswas ~80%,~80%, whereaswhereas heparinheparin so-sodium exhibited a 63% inhibition. diumdium exhibitedexhibited aa 63%63% inhibition.inhibition.

FigureFigure 3.3. Dose–responseDose–response relationships between the the hit hit compound compound concentrations concentrations and and RBD RBD-ACE2-ACE2 Figure 3. Dose–response relationships between the hit compound concentrations and RBD-ACE2 FigureFigure binding.binding.3. 3. Dose–response Dose–response InhibitionInhibition relationships relationships proﬁlesprofiles of of the the between between RBD-ACE2 RBD-ACE2 the the hi interactionhi interactiontt compound compound in in the concentrations thconcentrationse presence presence of of increasing and andincreasing RBD-ACE2 RBD-ACE2 concentrations concentra- binding.tions Inhibition of heparin profiles sodium of the (A RBD-ACE2), ATA (B), andinte ractionellagic acidin the (C presence), determined of increasing by the binding concentra- assay. The binding.binding.of Inhibition heparinInhibition sodium profiles profiles (A of of), the ATAthe RBD-ACE2 RBD-ACE2 (B), and ellagic inte interactionraction acid ( Cin in), the the determined presence presence byof of increasing theincreasing binding concentra- concentra- assay. The values tionstions ofofvalues heparinheparin are sodiumsodium the average ((AA),), ATA ATAof triplicates ((BB),), andand ± ellagicellagic SEM. acidTheacid IC((CC50),), valuesdetermineddetermined are listed byby thethe in bindingTablebinding 1. assay.assay. TheThe tions ofare heparin the average sodium of ( triplicatesA), ATA (B±),SEM. and ellagic The IC acidvalues (C), determined are listed in by Table the1 binding. assay. The values are the average of triplicates ± SEM. The IC50 values are listed in Table 1. valuesvalues are are the the average average of of triplicates triplicates ± ± SEM. SEM. The The IC IC5050 values values are are listed listed in in Table Table 1. 1. Table 1. RBD-ACE2 inhibition properties of the hit compounds. Table Table1. RBD-ACE2 1. RBD-ACE2 inhibition inhibition proper propertiesties of the ofhit the compounds. hit compounds. TableTable 1. 1. RBD-ACE2 RBD-ACE2 inhibition inhibition proper propertiesties of of the the hit hit compounds. compounds. IC50 Binding Inhibition Compound Structure ICIC50 50 BindingBinding Inhibition Inhibition CompoundCompound Structure Structure ICIC5050 [ µg/mL]BindingBinding InhibitionInhibition[%] CompoundCompound StructureStructure [µg/mL][µg/mL] [%] [%] [µg/mL][µg/mL] [%][%] heparin sodium 5.5 63 heparinheparinheparin sodiumsodium sodium 5.55.55.5 6363 63 heparin sodium 5.5 63

aurintricarboxylic acid (ATA) 0.6 80 aurintricarboxylic acid (ATA) 0.6 80 aurintricarboxylicaurintricarboxylicaurintricarboxylic acidacid acid (ATA)(ATA) (ATA) 0.60.60.6 8080 80

ellagic acid 2.5 84 ellagic acid 2.5 84 ellagicellagicellagic acidacid acid 2.52.52.5 8484 84

2.3. Evaluation of the Selected Compounds by a Plaque Reduction Assay 2.3.2.3. EvaluationEvaluation ofof thethe SelectedSelected CoCompoundsmpounds byby aa PlaquePlaque ReductionReduction AssayAssay 2.3. Evaluation2.3. EvaluationThe of efficacy the Selected of theof the Selected Co threempounds Compounds identified by a Plaque molecules, by a PlaqueReduction heparin Reduction Assay sodium, Assay ATA, and ellagic acid, TheThewas efficacyefficacy further ofof assessed thethe threethree by identifiedidentified a plaque reduction molecules,molecules, assay heparinheparin conducted sodium,sodium, in ATA,ATA, Vero andandE6 cells ellagicellagic using acid,acid, a SARS - The efficacyThe efﬁcacy of the three of the identified three identiﬁed molecules, molecules, heparin sodium, heparin ATA, sodium, and ellagic ATA, and acid, ellagic waswas furtherfurtherCoV-2 assessedassessed surrogate byby virus aa plaqueplaque (rVSV reductionreduction-SARS- CoVassaassay-y2 conductedconducted-S). The virus inin VeroVero was E6 E6preincubated cellscells usingusing aa with SARS-SARS- various was furtheracid, wasassessed further by a assessed plaque reduction by a plaque assa reductiony conducted assay in conductedVero E6 cells in using Vero E6a SARS- cells using CoV-2CoV-2concentrations surrogatesurrogate virusvirus of (rVSV-SARS-CoV-2-S).(rVSV-SARS-CoV-2-S). each molecule to ensure TheThe that virusvirus potential waswas preincubatedpreincubated interactions with withbetween variousvarious the com- CoV-2a surrogate SARS-CoV-2 virus surrogate (rVSV-SARS-CoV-2-S). virus (rVSV-SARS-CoV-2-S). The virus was The preincubated virus was preincubatedwith various with concentrationspounds and of each RBD molecule would occur to ensure prior thatto RBD potential-ACE2 interactionsinteraction. betweenThe mixtures the com- were then concentrationsconcentrationsvarious concentrations ofof eacheach moleculemolecule of each toto ensureensure molecule thatthat to potentialpotential ensure that interactionsinteractions potential interactionsbetweenbetween thethe between com-com- the poundsadded and RBDto cells would in six occur-well platesprior to for RBD-ACE2 72 h. The relativeinteraction. reduction The mixtures in plaque were formation then was poundspoundscompounds andand RBDRBD andwouldwould RBD occuroccur would priorprior occur toto priorRBD-ACE2RBD-ACE2 to RBD-ACE2 interaction.interaction. interaction. TheThe mixturesmixtures The mixtures werewere thenthen were then added to cells in six-well plates for 72 h. The relative reduction in plaque formation was addedaddedadded calculated, toto cellscells to inin cells six-wellandsix-well in IC six-well50 plates plateswas determined plates forfor 7272 for h.h. The 72The (Figure h. relativerelative The 4). relative reductionreductionATA reductionand inheparinin plaqueplaque in plaquesodium formationformation formation displayed waswas was po- calculated,calculated,tent antiviral andand ICIC5050 inhibition waswas determineddetermined activit ies (Figure(Figure in cells 4).4). (IC ATAATA50 values andand heparinheparin of 5.6 and sodiumsodium 73 µg displayeddisplayed/mL, respectively). po-po- calculated,calculated, and IC and50 was IC50 determinedwas determined (Figure (Figure 4). ATA4). ATA and and heparin heparin sodium sodium displayed displayed po- potent tenttent antiviralantiviralFor both inhibitioninhibition compounds, activitiesactivities the IC inin50 cellsvaluescells (IC(IC determined5050 valuesvalues ofof using 5.65.6 andand the 7373 plaque µg/mL,µg/mL, reduction respectively).respectively). assay were tent antiviralantiviral inhibition inhibition activities activities in in cells cells (IC (IC5050 valuesvalues of of 5.6 5.6 and and 73 73 µg/mL,µg/mL, respectively). respectively). For For both compounds, the IC50 values determined using the plaque reduction assay were ForFor bothbothbothan compounds,ordercompounds, compounds, of magnitude thethe the ICIC IC50 higher50 valuesvaluesvalues than determineddetermined determined those determined usingusing using thethe using the plaqueplaque plaque the reduction reductionbinding reduction assay, assayassay assay maintaining werewere were an an order of magnitude higher than50 those determined using the binding assay, maintaining anan order orderordertheir of of magnitude relativemagnitude of magnitude ranking. higher higher higher A than than dose than those those–response those det determinedermined determined curve using forusing ellagic using the the binding bindingacid the bindingcould assay, assay, not assay, maintaining maintaining be established maintaining , as their relative ranking. A dose–response curve for ellagic acid could not be established, as theirtheir relativerelativetheirconcentrations relative ranking.ranking. ranking. in AA the dose–responsedose–response range A dose–response of 1–120 curvecurve µg/mL fofo curverr ellagicofellagic this for compound acidacid ellagic couldcould acid notdidnot could benotbe established,established, inhibit not be rVSV established, asas-SARS - concentrations in the range of 1–120 µg/mL of this compound did not inhibit rVSV-SARS- concentrationsconcentrationsasCoV concentrations-2-S in infectionin the the range range inin theof Veroof 1–120 1–120 range E6 µg/mL cells.µg/mL of 1–120 These of of this µthis g/mLresults compound compound of demonstrate this did compounddid not not thatinhibit inhibit didATA rVSV-SARS- rVSV-SARS- not and inhibit heparin rVSV- so- CoV-2-S infection in Vero E6 cells. These results demonstrate that ATA and heparin so- CoV-2-SCoV-2-SSARS-CoV-2-Sdium infectioninfection are able inin infectionVerotoVero exert E6E6 potent cells. incells. Vero TheseThese inhibition E6 cells. resultsresults Theseof demonstratedemonstrateviral results infection, demonstrate thatthat presumably ATAATA and thatand heparinATAheparinby hindering and so-so- heparin the dium are able to exert potent inhibition of viral infection, presumably by hindering the diumdium sodiumarebindingare ableable are oftoto the exert ableexert spike to potentpotent exert to cellular inhibition potentinhibition ACE2. inhibition ofof viralToviral rule of infection,infection, viral out a infection, possible presumablypresumably reduction presumably byby hinderinginhindering plaque by hindering formation thethe the binding of the spike to cellular ACE2. To rule out a possible reduction in plaque formation bindingbindingbindingdue of of the tothe cytotoxicity, ofspikespike the to spiketo cellular cellular tothe cellular ACE2.effect ACE2. ACE2.of To To the rule rule compounds To out out rule a a possible possible out a at possible thereduction reduction highest reduction in in tested plaque plaque in concentrations plaque formation formation formation on due to cytotoxicity, the effect of the compounds at the highest tested concentrations on duedue totoduecell cytotoxicity,cytotoxicity, viability to cytotoxicity, was thethe evaluated effecteffect the effectofof the theusing of compoucompou the alamarBlue compoundsndsnds atat thereagent,the at highesthighest the by highest comparingtestedtested tested concentrationsconcentrations the concentrations viability onon of cells on cell viability was evaluated using alamarBlue reagent, by comparing the viability of cells cellcell viabilityviabilitycellincubated viability waswas with evaluatedevaluated was the evaluated compound usingusing usingalamarBluealamarBlue to the alamarBlue viability reagent,reagent, of reagent, cells byby comparingcomparing incubated by comparing theothenly viabilityviability with the viability media. ofof cellscells None of cells of incubated with the compound to the viability of cells incubated only with media. None of incubatedincubatedincubated with with the the with compound compound the compound to to the the toviability viability the viability of of cells cells of incubated incubated cells incubated only only with with only media. media. with media. None None Noneof of of the tested compounds were cytotoxic as the cell viability percentages following incubation with 120 µg/mL of ellagic acid, 169 µg/mL of ATA, and 650 µg/mL of heparin sodium

were 106%, 102%, and 97%, respectively. Molecules 2021, 26, x FOR PEER REVIEW 5 of 12

Molecules 2021, 26, 3213 the tested compounds were cytotoxic as the cell viability percentages following incubation5 of 12 with 120 µg/mL of ellagic acid, 169 µg/mL of ATA, and 650 µg/mL of heparin sodium were 106%, 102%, and 97%, respectively.

Figure 4. Inhibition of viral infection of cells by heparin sodium and ATA. rVSV-SARS-CoV-2-S Figure 4. Inhibition of viral infection of cells by heparin sodium and ATA. rVSV-SARS-CoV-2-S was was preincubated for 1 h with the indicated concentrations of heparin sodium (A) and ATA (B). preincubated for 1 h with the indicated concentrations of heparin sodium (A) and ATA (B). Mixtures Mixtures were then added to Vero E6 cells and incubated for 72 h. Cells were then stained using werecrystal then violet, added and to the Vero relative E6 cells plaque and incubated reductionfor was 72 determined. h. Cells were The then results stained are using expressed crystal as violet, the andpercent the relativeinhibition plaque relative reduction to that was of the determined. virus-only The control results incubated are expressed with the as thecorresponding percent inhibition sol- relativevent (infection to that ofmedium the virus-only without control or with incubated 2% DMS withO for the heparin corresponding sodium or solvent ATA, respectively). (infection medium The withoutvalues are or withpresented 2% DMSO as the for mean heparin of 6- sodiumwell replicates or ATA, ± respectively).SD. The lower The images values are are three presented representa- as the meantive wells of 6-well with replicatesthe indicated± SD. compound The lower concentrations. images are three representative wells with the indicated compound concentrations. 2.4. Molecular Docking Analysis 2.4. Molecular Docking Analysis Molecular docking was conducted to suggest a mechanism underlying the inhibition of theMolecular RBD-ACE2 docking interaction was conducted by ATA and to suggestheparin a sodium mechanism (Figure underlying 5). Molecular the inhibition docking ofwas the based RBD-ACE2 on the published interaction crystal by ATA structure and heparin of the sodiumRBD-ACE2 (Figure complex5). Molecular (resolution docking of 2.45 wasÅ ) [18] based. The on compounds the published were crystal allowed structure to freely of the dock RBD-ACE2 to the receptorcomplex binding(resolution motif of 2.45(RBM) Å)— [18the]. ACE2 The compounds binding site were within allowed the RBD to freely [19]. The dock RBM to the spans receptor amino binding acids 437 motif to (RBM)—the507 and forms ACE2 a concave binding surface site within that interacts the RBD with [19]. ACE2. The RBM The spanscompounds amino were acids docked 437 to 507to the and concave forms a surface concave of surface the RBM, that implying interacts withthat ACE2.their binding The compounds to the RBD were may docked disrupt to thethe concaveinteraction surface with ofACE2. the RBM, The calculated implying thatGibbs their free binding energy to(ΔG) the values RBD may for disruptthe interac- the interactiontion of the withcompounds ACE2. Thewith calculated the RBD Gibbswere free−8.9 energyand −7.05 (∆G) kCal values/mL forfor the heparin interaction sodium of the compounds with the RBD were −8.9 and −7.05 kCal/mL for heparin sodium and ATA, and ATA, respectively. The predicted binding site for heparin included Tyr505, which respectively. The predicted binding site for heparin included Tyr505, which participates in participates in the interaction of the RBD with ACE2. The predicted binding site for ATA the interaction of the RBD with ACE2. The predicted binding site for ATA was at a more was at a more pivotal location as, in addition to Tyr505, it includes Tyr449, Tyr453, Gln493, pivotal location as, in addition to Tyr505, it includes Tyr449, Tyr453, Gln493, Gln498, and Gln498, and Asn501, found at the RBD-ACE2 interface. Asn501, found at the RBD-ACE2 interface.

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FigureFigure 5. 5.A A dockingdocking modelmodel ofof thethe bindingbinding ofof heparinheparin sodiumsodium andand ATAATA toto thethe RBD.RBD. ((AA)) RBD-ACE2RBD-ACE2 interactioninteraction (pdb (pdb code code 6M0J). 6M0J). The The RBD RBD forms forms a concavea concave surface surface with with a protruding a protruding loop loop on oneon one side, side, to whichto which ACE2 ACE2 binds binds (ACE2 (ACE2 is colored is colored in light in bluelight andblue is and shown is shown in a ribbon in a ribbon presentation; presentation; RBD is RBD shown is inshown surface in (gold surface colored) (gold andcolored) ribbon and (red ribbon colored) (red representations). colored) representations). Heparin sodium Heparin (one sodium disaccharide (one unit)disaccharide and ATA unit) were and docked ATA to were the RBM docked using to the RBM SwissDock using serverthe SwissDock [20]. The server compounds [20]. The are showncom- pounds are shown in stick representation, where oxygen, sulfur, and hydrogen are colored in red, in stick representation, where oxygen, sulfur, and hydrogen are colored in red, yellow, and white, yellow, and white, respectively, and carbon atoms are colored in magenta for heparin sodium (B) respectively, and carbon atoms are colored in magenta for heparin sodium (B) and green for ATA (C). and green for ATA (C). The figure was prepared using UCSF Chimera [21]. The ﬁgure was prepared using UCSF Chimera [21].

3.3. DiscussionDiscussion TheThe globalglobal COVID-19COVID-19 pandemicpandemic ledled toto anan urgent urgent demand demand for for developing developing vaccines vaccines andand effectiveeffective treatments.treatments. InIn viewview ofof thisthis need,need, drugdrug repurposingrepurposing isis aa rapid rapid approach approach for for discoveringdiscovering beneficialbeneficial COVID-19COVID-19 therapeutics therapeutics by by using using established established drugs. drugs. Inhibiting Inhibiting the the RBD-ACE2RBD-ACE2 interaction,interaction, therebythereby preventingpreventing viralviral attachmentattachment toto targettarget cells,cells, isis usedused as as an an approachapproach toto develop develop vaccines vaccines and and therapeutics. therapeutics. TheThe latterlatter includeinclude antibodies,antibodies, aptamers,aptamers, peptides,peptides,and and specificspecific small-molecule small-molecule inhibitorsinhibitors (SMIs)(SMIs) [ 22[22––2828].]. One One effective effective and and rapid rapid strategystrategy for for discovering discovering inhibitors inhibitors is is by by screening screening compound compound libraries, libraries, as was as was recently recently ap- pliedapplied to find to find new new therapeutics therapeutics for COVID-19. for COVID For-19. example,For example, Gordon Gordon et al. et identified al. identified 332 new 332 SARS-CoV-2-humannew SARS-CoV-2-human protein–protein protein–protein interactions interactions and found and found two sets two of sets pharmacological of pharmaco- agentslogical that agents displayed that displayed antiviral antiviral activity [ 14activity]. In another [14]. In study, another the study, ReFRAME the ReFRAME (repurposing, (re- focusedpurposing, rescue, focused and accelerated rescue, and medchem) accelerated drug medchem) library was drug screened library for was inhibitors screened of for viral in- replication,hibitors of viral and 100replication, molecules, and including 100 molecules, 21 known including drugs, 21 were known found drugs, [13]. were However, found no[13] compound. However, was no compound associated withwas a blockingssociatedthe with RBD-ACE2 blocking the interaction RBD-ACE2 in eitherinteraction report. in Screeningeither report. for inhibitors, Screening particularly for inhibitors, SMIs, particularly that can potentially SMIs, that hamper can RBD-ACE2 potentially binding hamper hasRBD been-ACE2 reported binding in has several been in reported silico studies. in several Choudhary in silico studies. et al. identified Choudhary five et potential al. iden- SARS-CoV-2tified five potential cell entry SARS inhibitors-CoV-2 that cell wereentry shown inhibitors to virtually that were bind shown the spike to virtually protein [bind15]. Wuthe etspike al. protein analyzed [15] therapeutic. Wu et al. analyzed targets in therapeutic SARS-CoV-2 targets and in virtually SARS-CoV screened-2 and virtually several compoundscreened several databases compound for the inhibitiondatabases offor these the inhibition targets. One of these compound targets. (hesperidin) One compound was suggested(hesperidin) to bindwas suggested the binding to interfacebind the betweenbinding theinterface spike between and ACE2 the [17 spike]. In and the studyACE2 of[17] Wang. In the et al.,study eight of Wang common et al., types eight of common potential types inhibitor of potential structures inhibitor wereoutlined structures by were ap- plyingoutlined a computational by applying a computational screening strategy screening based strategy on blocking based S RBD/hACE2 on blocking S binding RBD/hACE2 [16]. Despitebinding the [16] advantages. Despite the of the advantages in silico screening of the in approach, silico screening the in vitro approach,screening the ofindrug vitro librariesscreening can of add drug a functionallibraries can level add to a the functional process, level further to promotingthe process, the further discovery promoting of potent the inhibitorsdiscovery of of the potent RBD-ACE2 inhibitors interaction. of the RBD-ACE2 interaction. InIn the the current current study, study, a newa new binding binding assay assay was was developed developed and usedand used to screen to screen two drug two librariesdrug libraries for inhibitors for inhibitors that interfere that interfere with the with RBD-ACE2 the RBD interaction.-ACE2 interaction. Three compounds Three com- thatpounds inhibited that inhibited over 60% over of the 60% interaction of the interaction were selected. were selected. Two compounds, Two compounds, heparin sodium heparin and ellagic acid, are FDA-approved drugs. In addition to its anticoagulant activity, hep- sodium and ellagic acid, are FDA-approved drugs. In addition to its anticoagulant activ- arin has previously been found to inhibit infection by several viruses, including human ity, heparin has previously been found to inhibit infection by several viruses, including immunodeficiency virus (HIV), coronavirus NL63, and Zika virus [29–31]. In line with human immunodeficiency virus (HIV), coronavirus NL63, and Zika virus [29–31]. In line our screening results, it has recently been shown that the spike protein of SARS-CoV-2 with our screening results, it has recently been shown that the spike protein of SARS-CoV-

Molecules 2021, 26, 3213 7 of 12

strongly binds immobilized heparin [32]. A docking analysis suggested putative heparin (and other glycosaminoglycans) binding motifs in the RBD when it is in an open conformation [32]. Interestingly, Clausen et al. [33] provided evidence that heparan sulfate, a cell membrane glycosaminoglycan, shifts the RBD to an open conformation to facilitate ACE2 binding. Additionally, it was suggested in their work that heparin could inhibit the role of heparan sulfate as a coreceptor for SARS-CoV-2 infection [33]. Most recently, heparin was found to inhibit SARS-CoV-2 infection in Vero cells [34]. The fact that heparin was specifically identified by the screening assay as a potent inhibitor strongly supports the reliability of the assay developed and applied herein for discovering effective compounds capable of directly blocking the RBD-ACE2 interaction. It is important to note that in an unrelated manner to SARS-CoV-2 inhibition, heparin is administered as an anticoagulant to COVID-19 patients suffering from coagulopathy and thromboembolism [35]. Thus, it appears that heparin is able to exert a beneficial synergistic effect in treating COVID-19 patients based on two distinct mechanisms. The third inhibiting compound is ATA, a polyanionic aromatic compound, which is recognized as a potent DNA topoisomerase II inhibitor [36]. Additionally, ATA exhibits inhibitory properties against several viruses and bacteria. Interestingly, among the viruses is the SARS coronavirus [37]. Other pathogens include HIV-1 [38], influenza virus [39], Vaccinia [40], Zika virus [41], and Yersinia pestis [42]. Ellagic acid, a polyphenol, is an investigational drug used to treat cancer, cardiovascular disease, and brain injury [43–54]. Similar to heparin and ATA, ellagic acid is also associated with antiviral activity against various viruses [55–57]. Recently, ellagic acid was identified by network analysis and molecular docking as a possible modulator of casein kinase II subunit alpha, a host protein that may be involved in the pathology of SARS-CoV-2 [58]. The three hit compounds were further characterized for their ability to inhibit the infection of Vero E6 cells by a pseudotyped virus expressing the SARS-CoV-2 spike protein using a plaque reduction assay. Only ATA and heparin sodium preserved their inhibitory activity in the cellular system. The IC50 values obtained in the cellular assay were an order of magnitude higher than those obtained in the binding assay, and in both assays, ATA demonstrated a higher inhibitory potency. Of note, the IC50 value obtained herein for heparin sodium is in line with that described in a recent report for the inhibition of the SARS-CoV-2 virus [34]. The ranking of IC50 values of ATA and heparin coincides with the results of the docking analysis. The higher potency of ATA can be explained in light of the prediction that ATA binds multiple residues of RBD at the interface of the RBD-ACE2 interaction, whereas heparin binds only one residue. The docking analysis may also serve to predict the binding of the identified compounds to SARS-CoV-2 variants. Of note, it has been reported that Asn501 is a critical residue for ACE2 binding by the RBM of SARS-like viruses [19]. This residue, according to the docking model, is part of the binding site of ATA. In this regard, several SARS-CoV-2 variants of concern listed by the CDC [59] (B.1.1.7, B.1.351, and P.1) contain an Asn501Tyr mutation. Although it can be hypothesized that a mutation in Asn501 might alter the binding of ATA to the RBD, the impact of the Asn501Tyr mutation on the potency of ATA is yet unknown. In the case of heparin, the docking analysis predicted that the binding site does not include residues mutated in the SARS-CoV-2 variants of concern. This suggests that the heparin binding to RBD of SARS-CoV-2 variants may not be altered. No effective inhibition of the rVSV-SARS-CoV-2-S virus could be demonstrated in Vero E6 cells by ellagic acid. It should be noted that the elimination of hit compounds between the primary and secondary screens is a characteristic of the screening funnel. However, we cannot rule out the possibility that, although ineffective in cells, ellagic acid could still be potent in vivo. Eubanks et al., for example, reported that two botulinum toxin inhibitors were effective in vivo even though they were ineffective in a cellular assay [60]. Further investigation is required to determine the inhibitory activity of ellagic acid against SARS-CoV-2. Molecules 2021, 26, 3213 8 of 12

In the absence of a specific anti-SARS-CoV-2 therapy, repurposing approved drugs may be useful for treating COVID-19 [11,61]. The current study can expand the limited drug toolkit by identifying approved drugs and pharmaceutically active compounds that can potentially be repurposed to inhibit the viral cell attachment step. The results provide a basis for further research with SARS-CoV-2 to evaluate the efficacy of hit compounds. Future structure–activity research (SAR) may allow one to design modifications that en- hance the affinity of the compounds, required to achieve improved therapeutic efficacy and selectivity. Interestingly, as these drugs have been reported to counteract additional viruses as well, they may serve as broad-spectrum antiviral therapeutics. These drugs can also be considered for use in a combined administration to increase the treatment efficacy, which may be further advantageous as their doses in a combined regimen can be reduced, thus minimizing the risk of side effects. Moreover, the binding assay developed herein can be applied for the high-throughput screening of additional compound libraries to facilitate the discovery of additional potent compounds.

4. Materials and Methods 4.1. Expression and Puriﬁcation of ACE2 and Fc-RBD A synthetic gene encoding the peptidase domain of Homo sapiens ACE2 (amino acids 19-615, GenBank: BAB40370.1), with optimized codon usage for expression in Escherichia coli and a C-terminal His-tag, was prepared by GenScript (Piscataway, NJ, USA) and cloned into pET-9a between the NdeI and BamHI sites. The plasmid pET-9a-ace2 was transformed into E. coli BL21(DE3). To express the protein, E. coli BL21(DE3)+pET-9a-ace2 was grown at 18 ◦C in TB media and was harvested after 40 h of incubation. To purify the protein, the cell pellet was resuspended in binding buffer (20 mM of sodium phosphate, 0.5 M of NaCl, and 20 mM of imidazole; pH 7.4; 100 mL per 25 g of wet cells) and disrupted by three passages in a cell homogenizer at 25 kPa (C series cell disruptor 1.1 KW, Constant Systems). The cell extract was centrifuged (14,000 g, 30 min) and the supernatant was loaded onto a 5 mL HisTrap FF column (GE Healthcare, Chicago, IL, USA) mounted on an AKTA Explorer FPLC system (GE Healthcare). The column was washed with 10 CV of binding buffer and 10 CV of binding buffer containing 40 mM of imidazole. The protein was eluted from the column with elution buffer (20 mM of sodium phosphate, 0.5 M of NaCl, 500 mM of Imidazole, pH 7.4). The puriﬁed protein was dialyzed against PBS and stored at −70 ◦C. The recombinant hFc-RBD fusion protein, expressed as recently described [22], was kindly provided by Dr. Ohad Mazor Lab (IIBR, Ness Ziona, Israel).

4.2. RBD-ACE2 Binding Assay Ninety-six-well plates (Maxisorp, Nunclon) were coated with ACE2 (100 ng/well) for 1 h at 37 ◦C. The plates were blocked for 1 h at 37 ◦C with TSTA buffer (2% (w/v) bovine serum albumin (BSA), 0.9% NaCl, 0.05% Tween 20, 50 mM of Tris, pH 7.6). For initial screening, a concentration of 10 µM of each compound from the LOPAC®1280 (Sigma, St. Louis, MO, USA) and DiscoveryProbeTM FDA-approved drug (APExBIO) libraries was incubated with 1 µg/mL of Fc-RBD in a total reaction mixture volume of 50 µL. For the IC50 experiments, 3-fold serial dilutions of the indicated compound (1 mM to 5.6 nM) were incubated with Fc-RBD (1 µg/mL). The mixtures (50 µL) were incubated for 1 h at 25 ◦C. Following incubation, the mixtures were added to the ACE2-coated plates and incubated for 1 h at 37 ◦C. After washing, the plates were incubated for 1 h at 37 ◦C with that alkaline phosphatase-conjugated goat anti-human Fc fragment (Sigma, St. Louis, MO, USA), diluted 1:1000 in blocking solution. The plates were washed, and the colorimetric reaction was developed using p-nitrophenyl phosphate (pNPP, Sigma, St. Louis, MO, USA). The absorbance was measured at 405 nm with a Synergy HTX ELISA reader (Biotek, USA). The assay included a rabbit anti-SARS-CoV-2 RBD polyclonal antibody as a positive control. The antibody was kindly provided by Dr. Ohad Mazor Lab (IIBR, Ness Ziona, Israel). Naïve rabbit serum served as a negative control. The IC50 values were calculated Molecules 2021, 26, 3213 9 of 12

using a 4-parameter logistic ﬁt by Gen5 software (Biotek, Winooski, VT, USA). The z-prime parameter for each plate was calculated by Gen5 software.

4.3. Plaque Reduction Assay The assay was conducted in Vero E6 cell monolayers (ATCC® CRL-1586™). The cells were grown in growth medium (Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 1% MEM nonessential amino acids (NEAA), 2 mM of L-glutamine, and 0.5% Pen/Strep, all from Biological Industries, Kibbutz Beit-Haemek, ◦ Israel) and were cultured at 37 C and 5% CO2. The cells were infected with 60 PFU/well of rVSV-SARS-CoV-2-S, which served as a SARS-CoV-2 surrogate virus. This virus is a replication-competent recombinant VSV-∆G-spike virus in which the glycoprotein of VSV (vesicular stomatitis virus) is replaced by the spike protein of SARS-CoV-2 [62]. Cells were seeded in 6-well plates (7 × 105 cells/well) and grown overnight in growth medium. A fixed dose (600 PFU/mL) of rVSV-SARS-CoV-2-S was incubated with different inhibitor concentrations of ATA, ellagic acid, and heparin (1:1, v/v) in infection medium (MEM containing 2% FBS with 1% NEAA, 2 mM of glutamine, and 0.5% P/S) and was used to infect Vero E6 monolayers in six replicates (200 µL/well). After incubation for 1 h at 37 ◦C, 3 mL/well of overlay (MEM containing 2% FBS and 0.4% tragacanth (Merck, Herzliya Pituach, Israel)) was added to each well, and the plates were incubated at 37 ◦C and 5% CO2 for 72 h. The media were then aspirated, and the cells were fixed and stained with 3 mL/well of crystal violet solution (Biological Industries, Kibbutz Beit-Haemek, Israel). The number of plaques in each well was determined, and the reduction in infection was calculated relative to the control wells containing no compound. The IC50 values were calculated using GraphPad Prism.

4.4. Cytotoxicity assaY A cytotoxicity assay was performed in Vero E6 cell monolayers using the alamarBlue cell viability reagent (Thermo Fisher Scientific). Cells (2 × 104 cells/well in 100 µL) were seeded in 96-well plates. After an overnight incubation at 37 ◦C, 80 µL of medium was aspirated, and 20 µL of inhibitors was added in triplicate to the wells. After 1 h of incubation, 20 µL was aspirated, 150 µL of overlay was added, and the cells were incubated at 37 ◦C for three days. Then, 19 µL of alamarBlue reagent was added per well and incubated for 2.5 h at 37 ◦C. The fluorescence results were determined by excitation at 530 nm and the collection of emission at 580 nm was determined using a CLARIOstar® reader (BMG labtech, Ortenberg, Germany). The cell viability was calculated by dividing the fluorescence results of the compound-containing wells by those of the control wells (containing no compounds).

4.5. Molecular Docking Analysis Molecular docking of the selected compounds was performed using the SwissDock server (www.swissdock.ch accessed on 7 October 2020 [20]). The RBD of pdb ﬁle 6M0J [18] was set as a target, and the 3D structures of the hits were from the ZINC database [63].

Author Contributions: Conceptualization, A.B.D., E.D. (Eran Diamant), R.Z. and A.T.; methodology, A.B.D., E.D. (Eran Diamant), R.Z. and A.T.; investigation, A.B.D., E.D. (Eran Diamant), E.D. (Eyal Dor), A.B., N.N., L.L., S.C., L.C.M., E.E. and A.T.; writing—original draft preparation, A.B.D., E.D. (Eran Diamant) and A.T.; writing—review and editing, R.Z. and A.T. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Israel Institute for Biological Chemical and Environmental Sciences (SB182). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Molecules 2021, 26, 3213 10 of 12

Data Availability Statement: The data presented in this study are available on request from the corresponding authors. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Sample Availability: Samples of the compounds are available from the authors.

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