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Preprint:Pleasenotethatthisarticlehasnotcompletedpeerreview.

ComputationalscreeningapproachesforinvestigatingpotentialactivityofphytoligandsagainstSARS-CoV-2CURRENTSTATUS:POSTED

AcharyaBalkrishnaPatanjaliResearchInstitute

PallaviThakurPatanjaliResearchInstitute

ShivamSinghPatanjaliResearchInstitute

SwamiNarsinghPatanjaliResearchInstitute

NamitaSinghGuruJambheshwarUniversityofScienceandTechnology

RakeshKumarSharmaSaveethaInstituteofMedicalandTechnicalSciences

rksharmadrl@yahoo.comCorrespondingAuthor

DOI:10.21203/rs.3.rs-23206/v1

SUBJECTAREASBioinformatics NaturalProductChemistry

KEYWORDSCoronavirus,COVID-19,Drugdesigning,Herbaldrug,Moleculardocking,SARS-CoV-2

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AbstractObjective:SARS-CoV-2causesCOVID-19,alife-threateningrespiratoryillnesswithhighratesof

morbidityandmortality.Asondate,thereisnospecificmedicinetopreventortreatCOVID-19.

Therefore,thereisanacuteneedtoidentifyevidence-basedholisticandsafemitigators.

Methods:Thepresentstudyisaimedtoscreenligandsofherbaloriginusingrationalebased

bioprospectionanalysisandsubsequentlypredicttheirbindingpotentialsubduethemajordrug

targetsfornovelCoronavirusbyemployingcomputer-aidedvirtualscreening.Further,comparative

analysisofthebindingpotentialofanapprovedchemicalanalogueandselectedherballigandswere

alsopredicted.Theselectionofreceptorswasperformedbasedontheirpathophysiologicalrelevance,

asassessedbyaPubMedbasedkeywordhitsmatrixanalysis.ThedruglikelinessandADMETox

descriptorsof24herballigandswerecomputationallypredicted.Dockingstudieswerefurther

conductedwiththosephytoligandsthatqualifiedtheseparameters.Anexistingantimalarialdrug,

hydroxychloroquine,wasalsodockedwithalltheselectedviralreceptorsanditstheoreticalbinding

energywassetupasastandardforcomparisonaswellasscrutinizationofbindingenergiesofthe

phytoligands.

Results:Thedockingstudiessuggestedthattheherballigand,namely,gamma-glutamyl-S-

allylcysteinedemonstratedhighlysignificantbindingenergieswithviralspikeglycoprotein,

endoribonucleaseandmainprotease(bindingenergy≥-490kcal/molforallthetestedviral

receptors).

Conclusion:Gamma-glutamyl-S-allylcysteinedemonstratedmoresignificantbindingpotentialas

comparedtotheknownchemicalanalogue,i.e.,hydroxychloroquine,asobservedinthe

computationaldockingstudies.Thisstudyservestopresentpre-eminentinformationforfurther

clinicalstudieshighlightingtheutilityofherballigandsasprobableleadmoleculesformitigating

novelCoronavirusinfection.

1.IntroductionCoronaviruses(CoVs)representacategoryofinfectiousagents,belongingtothefamily

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Coronaviridae,categorizedintofourgenera,namely,alpha-CoV,beta-CoV,gamma-CoV,anddelta-

CoValongwiththeirsubclasses(Lefkowitzetal.,2018).TheseCoVscontainlargehost-derived

membraneenvelope,helicalnucleocapsid,non-structuralproteins(nsp1–16)andpositivesense,

single-strandedRNA(~26–32kilobases).TheviralRNAiscapableenoughofinitiatinginfectionwithin

thehost,evenwithoutthepresenceofessentialenzymes(Tripp&Tompkins,2018).Sincethe

beginningofthe21stcentury,CoVshavecausedthreezoonoticoutbursts,namely,severeacute

respiratorysyndrome(SARS-CoV;2002–2003),MiddleEastrespiratorysyndrome(MERS-CoV;2012),

andtheCOVID–19,causedbysevereacuterespiratorysyndromeCoronavirus2(SARS-CoV–2;2019)

(Zakietal.,2012;Ouetal.,2020).ThenovelCOVID–19pandemicoriginatedfromtheHubeiprovince

ofCentralChinaduringthelateNovember,2020,withitsepicenterbeinginWuhancityharbouring

nearly11millionpeople(Huetal.,2020).Thisinfectionhasbeengrowingsincethenandhasspread

tomorethan205countries(Cucinotta&Vanelli,2020;WHO,2020).Thetransmissibilityand

penetrancerateofthisinfectionisfrequentlychangingathourlyanddailybasis(Roseretal.,

2020).TheclinicalmanifestationsofthenovelCOVID–19infectionincluderespiratory,gastrointestinal,

nephrologicalandneurologicaletiologies(Balkrishnaetal.,2020).Primarytransmissionofthis

infectioninvolvedzoonoticspreadfromanimals(possiblybats)tohumans(Zhangetal.,2020;Zhou

etal.,2020).Inparticular,theprimarytransmissionofCOVID–19infectionwasapparentlylinkedto

theingestionofliveanimalandseafood(Balkrishnaetal.,2020).AlthoughthebatCoVbears

maximumsimilaritywithSARS-CoV–2,butduetothedifferenceinspecificaminoacidresidues,the

batCoVcouldnotdirectlyinfecthumans,neverthelesstherewouldbeanintermediatehostbetween

batsandhumansforthetransmittanceofthevirus(Cyranoski,2020).Subsequently,secondary

transmissionoccurredamonghumans,leadingtoafasterrateofspreadoftheinfectionwithinthe

community(Guanetal.,2003;Balkrishnaetal.,2020).TheincubationperiodofSARS-CoV–2ranges

between1–14dayswiththemedianincubationperiodofabout5days.Aninfectedpersonwill

apparentlyshowsymptomsafterthemedianincubationperiod.However,theinfectedindividualcan

spreadtheinfectionsincecontractingthedisease.TheprimarymodeoftransmissionofCOVID–19

infectionisthroughcontactwiththeinfectedindividualsand/orbyrespiratorydroplets(Carlosetal.,

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2020).SomestudieshavealsoproposedthatSARS-CoV–2mayalsopropagatebythefaecal

shreddingofinfectedorevencuredindividuals(Yeoetal.,2020).

TheCOVID–19infectionhasbecomeaglobalhealthchallengeowingtoitshighmorbidity(~3.17%);

transmissionrate(~2.2perpatient)(Sunetal.,2020);longerhalf-lifeofvirus(~4–72hours);and

asymptomaticmodeoftransmission(~2–14daysofincubation)(Huangetal.,2020;Balkrishnaetal.,

2020).ThemostcommonindicationsofCOVID–19compriseoffever,nasopharyngealcongestion,dry

cough,sorethroat,dyspnoea,diarrhoea,neurologicaldisordersandmultipleorganfailure.However,

somepatientsactasasymptomaticcarriersofthediseasewhicharemostdifficulttodiagnoseand

consequentlytreat.Eventhoughthedistinctsymptomsseemtobemild,butcanrapidlyenhanceto

severeconditions,andevendeath(Huangetal.,2020;Kim,2020).

TheexactmolecularpathogenesisofSARS-CoV–2isyetnotknownwithcertainty;however,ithas

beenproposedthatviralpathogenesisistriggeredbythereleaseofproinflammatorycytokinesthat

areassociatedwiththeactivationofseveralsignalingpathways,namely,TLRs-dependentIFN

inductionpathways(IRF–3/7&NF-Kβ)andmyeloid-differentiationprimaryresponse88pathways(ATF–

2&AP–1).TheprimaryvirulencefactorsofthevirusincludeSARS-CoV–2spikeglycoprotein(viral

envelopeproteinresponsibleforviralattachmentandentrywithinthehostcells);viralnuclease

(NSP15endoribonucleaseresponsibleformediatingviralcapsidformation);andprotease(Main

Protease3CLproresponsibleforviralcapsidformation)(Chenetal.,2020).SARS-CoV–2mainly

targetsthealveolarandbronchialepithelialcells.TheviralspikeglycoproteininteractswiththeACE–

2receptorsofthehostcell,therebymediatingtheviralentry.Afterwards,thereoccursanelevated

releaseofproinflammatorycytokines(IL–6andIL–12),chemokines(IL–8,CCL–2andCXCL10)and

interferon.Thevirusgetsholdofthehostcellmachineryandmanipulatesitfordrivingtheprocessof

viralreplication.Virusalsoattemptstoinhibittheproductionofinterferonandproinflammatory

cytokinesbymeansofRNAhelicasesandnon-structuralproteins(Nsp1,3,7&15).Subsequently,

theviralloadkeepsonrisingwithinthehostsystem,ultimatelyleadingtoviraemia(Figure1)..

Meanwhile,thehostimmunesystemalsostrivestofightback.Thefurtherspreadofviruswithinthe

hostsystemdependsontheimmunologicalstatusofthehost.Immunomodulatoryremediesplaya

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pivotalroleatthisphase,whereinsuchmoietiesmaymediatethetransformationofan

immunocompromisedindividualtoanimmunocompetentone.

Although,thestructureandsequenceofSARS-CoV–2havebeenidentifiedanddrugscreening

followedbyclinicaltrialsarecontinuouslybeingconductedbytargetingthesevirulencefactors.

However,therearenoapproveddrugsforeffectivelymanagingCOVID–19infection,probablydueto

unidentifieddynamicpathophysiology;highmutagenicityofthevirus;andadversesideeffectsof

earlierknownCoronavirusvaccinesanddrugs(Guoetal.,2020;Senathilakeetal.,2020;Wangetal.,

2020).Inlightoftheoutbreak,severaltreatmentregimeshavebeenproposed,includingtraditional

medicinalmoieties,thathavebeenextensivelyusedduringtheformerepidemicoutbreaks,suchas

SARSandH1N1influenza(Zakaryanetal.,2017;Guerrieroetal.,2018;Yangetal.,2018;Angetal.,

2020;Joetal.,2020).Herbalsprovideauniquesolutionintermsoftheirnegligiblesideeffects,

synergisticactivity,broadspectrumtherapeuticabilityandimmunomodulationeffects(Thakuretal.,

2016).

Inthepresentstudy,thedrug-likepotentialofsecondarymetabolitesofherbaloriginwillbestudied

bytargetingSARS-CoV–2spikeglycoprotein(S2),viralnuclease(NSP15endoribonuclease)and

protease(MainProtease3CLpro).Theselectionofherbalsecondarymetabolitesisbasedon

investigatingthepotencyofvariousherbalsshowingresistancetowardsviralvirulencefactorsas

indicatedbyvariousscientificsearchenginesbasedonpriorityindexing.Alltheselectedherbal

moietiesweresubjectedtomoleculardockingusingHexsoftware,soastoassesstheinteractionsof

phytoligandswiththeviralreceptors.Furtherscreeningofthepropitiousherballeadswasconducted

byassessingtheinsilicotoxicityandLipinskiscorebaseddruglikeliness.Bulkoutliersshowinghigh

toxicity/mutagenicityorviolatingtheLipinskiruleswereeliminated.Subsequently,thedrug-receptor

interactionofthefilteredherbalmoietieswasstudiedsoastoobtainaleadmoleculewhichcouldbe

furthertestedatpreclinicalandclinicallevels.Althoughsearchforpotentialleadstargetingthenovel

Coronaviruswillcontinueperpetually,buttheseherballeadsmayservetobehighlybeneficialowing

totheirantiviralactivities,potentiatingnatureandsymptomaticreliefprovisioncapabilities,

presentedalongwithlimitedtoxicitiesandcomprehensivetreatmentstrategy.

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2.MaterialsAndMethods2.1Selectionandpreparationofviralvirulencefactorsasreceptors

ThereceptorswereselectedbasedontheirroleincausingviralinfectionbySARS-CoV–2virus.These

receptorswereassignedaweightagescoreonthebasisoftheirpathophysiologicalrelevance.Such

pertinencewasassessedbyconductingakeywordhitsscoringmatrixusingPubMedsearchengine.

Therandomkeywordsearchwasconductedusingcombinationkeywordas‘virulencefactorinhibition

+antiviralactivity’.Thefirst20hitsprovidedbythesearchengine,workingontheprincipleof

priorityindexing,werebasedonthenumberoftimesawebsiteisclicked(Qinetal.,2005).This

samplesetbasedanalysiswasusedtoevaluatethenetweightagelinkedtoeachvirulencefactor,

usingthefollowingformula:

Thecrystalstructuresoftheselectedrelevantproteintargets,namely,SARS-CoV–2spike

glycoprotein(S2;PDBcode:6VSB),viralnuclease(NSP15endoribonuclease;PDBcode:6VWW)and

protease(MainProtease3CLpro;PDBcode:1Q2W)wereobtainedfromRCSBProteinDataBank

(https://www.rcsb.org/).ThesestructureswereexaminedcriticallyusingRamachandranPlotby

ProChecksoastovalidatethemodeledproteinstructuresbasedontheφ(phi),ψ(psi)andω(omega)

angles,therebyinspectingthequalityofthetargetproteinstructuresselectedfordockingstudies.

Furthermore,hydrogenatomswereintroducedinallthese3DstructuresusingArgusLab(4.0.1),so

astocustomizetheviralreceptorsforrigiddocking

(http://www.arguslab.com/arguslab.com/ArgusLab.html).

2.2Activesiteanalysisofviralvirulencefactors

ThepredictionofactivesitesofviralreceptorswasaccomplishedbyDoGSiteScorer,andthe

Cartesiancoordinatesx,y,z(activesites)foreffectivedockingwerevisualizedinArgusLab.These

regionswerefurtherusedforthegenerationofgridboxfordockingstudiesbyHexCuda8.0.0.

PredictionswithDoGSiteScorerwerebasedonthedifferenceofGaussianfiltertodetectpotential

pocketsontheproteinsurfacesandtherebysplittingthemintovarioussub-pockets.Subsequently,

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globalproperties,describingthesize,shapeandchemicalfeaturesofthepredictedpocketswere

calculatedsoastoestimatesimplescoreforeachpocket,basedonalinearcombinationofthree

descriptors,i.e.,,volume,hydrophobicityandenclosure.Foreachqueriedinputstructure,a

druggabilityscorebetween0-to–1wasobtained.Higherthedruggabilityscore,higherthe

physiologicalrelevanceofthepocketaspotentialtarget.Theactivesiteswithbestdruggability

scoreswereenvisagedasanessentialrequirementforthecreationofgridinselectedviralreceptors

(Volkameretal.,2012).

2.3Selectionandpreparationofherbalsaspromisinganti-SARS-CoV–2candidates

Atotalof24bioactivecompoundsfromnaturallyavailablemedicinalplantswereselectedby

employingabiostatisticalmatrixbasedanalysis.Basedontheunderstandingofthe

pathophysiologicaltargetsofthenovelCoronavirus,herbalcandidatesexhibitinginhibitoryproperties

specifictotheviralvirulencefactorsweresearchedinthePubMedrepository.Thedescriptorsused

forconductingPubMedsearchwiththehelpofextensiveliteraturesurgeincludedkeywordsas

‘virulencefactorinhibition+herbalmoiety’.Binarycoefficientfortheherbalmoietieswerecalculated

byassessingthepresenceorabsenceofparticularinhibitingpropertyexhibitedagainstindividual

physiologicaltarget.Thebinaryscoreforeachplantrangedfrom0to6,whereinthemediancut-off

valuewasselectedas3.Plantshavingabinaryscore≥3wereconsideredforfurtherweightage

basedmatrixanalysis,whereinthebinaryscoreofeachplantwasmultipliedwiththerelevancescore

oftheviralvirulencefactor(RefertoSection2.1).Ultimately,afuzzysetmembershipanalysiswas

conductedinordertoobtainauniversalscoreforeachplant.Thefuzzysetscorerangedbetween0

and1,whereintheplantswithafuzzyscoregreaterthan0.5werefurtherselectedforassessingtheir

specificanti-SARS-CoV–2activity.

where,μSrepresentsthedesirabilityvaluesofmembersofthefuzzysetS;min(S)andmax(S)are

minimumandmaximumvalues,respectively,inthefuzzysetS.

Predominantphytochemicaloftheselectedplants(withfuzzyscore≥0.5)wereidentifiedby

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extensiveliteraturesurge.Thethree-dimensionalstructuresofallthesebioactivemoleculesaswell

asthereferencedrugcompound(hydroxychloroquinewhichisunderconsiderationasaremedyfor

novelCOVID–19viraldisease)wereretrievedfromPubChemdatabase.Hydrogenatomswere

introducedintotheligandsstructureusingArguslab(4.0.1),soastocustomizethemforrigid

docking.ThehydrogenatedligandmoleculeswerethenconvertedintopdbformatusingOpenBabel

(2.4)interface(openbabel.org/docs/dev/OpenBabel.pdf),asrequiredforrigiddocking.Similarly,3D

structureofstandardchemotherapeuticagent(hydroxychloroquine)wasalsocustomizedfordocking

(Modietal.,2013;Chakotiyaetal.,2014).

2.4Insilicopharmacokineticanalysis

2.4.1DrugLikeliness

Druglikelinessoftheselectedphytoligands(~17compounds)wasassessedbyusingDruglikeness

toolDruLiTo,whichisanopensourcevirtualscreeningtoolforcalculatingLipinski’sruleoffive,i.e.,

molecularweight,numberofhydrogenbonddonors,numberofhydrogenbondacceptorsandLogP

value.Violationofmorethanonerulewouldcauseexclusionofthesaidphytochemical.Restofthe

selectedphytoligandsweresubjectedtoADMEToxanalysis(Kriszhnamurthietal.,2018)

2.4.2ADMEToxAnalysis

TheADMETox(Absorption,Distribution,Metabolism,ExcretionandToxicity)descriptorsofthe

selectedphytocompoundswerepredictedbyconductingadmetSAR.Theestimationoftheprobability

valuesofthecompoundsfordiverseprofilesincludinghumanoralbioavailability,humanepithelial

colorectaladenocarcinomacell(CaCo2)permeability,logPforsubstratesandinhibitors,predicted

aqueoussolubilityanddifferenttoxicityprofilesintermsofAMEStoxicityandoraltoxicity(LC50and

LD50values)werecomputationallypredicted.Phytomoleculesexhibitingtoxicprofileasassessedin

theinsilicotoxicityanalyseswereexcludedfromthestudy.Restoftheselectedphytoligandswere

subjectedtomoleculardockinganalysis(Nishaetal.,2016).

2.5MolecularDockingandLigandReceptorBindinganalysis

Thedockinganalysisofpdbstructuresofselectedphytoligands(excludingtheLipinskiruleand

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ADMEToxviolatingmoieties)withviralreceptors(spikeglycoprotein,viralnucleaseandviralmain

protease)wascarriedbyHexCuda8.0.0software.ReceptorandLigandfileswereimportedinthe

software.ThegriddimensionofdockingwasdefinedaccordingthetothebindingsiteanalysisofDoG

SiteScorer.GraphicsettingsandDockingparameterswerecustomizedsoastocalculatethebinding

energies(Evalues)ofligandreceptordocking.Theparametersusedforthedockingprocesswereset

as(i)Correlationtype:Shape+Electro+DARS,(ii)FFTmode:3Dfastlite,(iii)GridDimension:0.7,

(iv)Receptorrange:180°,(v)Ligandrange:180°,(vi)Twistrange:360°.Thebestdocked

conformationswithlowestdockingenergywereselectedforfurtherMDsimulationsusingPoseView

forcreatingposedepictionsofselectedligand-receptorbinding(Harikaetal.,2017).

3.ResultsAndDiscussion3.1Selectionofviralvirulencefactors

Onthebasisofthekeywordhitsscoringresults,weightagewasgiventovariousparametersselected

forscreeningofherballeadswithrespecttoantiviralactivity.Weightagewasdecidedaccordingto

thepercentagerelevanceobtainedforeachparameter,asgiveninTable1.Highestpercentage

relevancewasobtainedforviralspikeglycoproteininhibitor,followedbyotherparameterslikeviral

endonucleaseinhibitorandviralproteaseinhibitor.Dockingstudieswerehenceperformedwiththese

viralreceptors,namely,SARS-CoV–2spikeglycoprotein(S2),viralnuclease(NSP15endoribonuclease)

andprotease(MainProtease3CLpro).

3.2Qualityassessmentofviralvirulencefactors

Thefirstqualityassessmentoftheselectedviralvirulencefactors(SARS-CoV–2spikeglycoproteinS2,

viralNSP15endoribonucleaseandmainprotease3CLpro)wascarriedoutusingRamachandranplot

analysiscomputedwithProCheck.TheanalysisshowedthatresiduesofSARS-CoV–2spike

glycoproteinS2,viralNSP15endoribonucleaseandmainprotease3CLprointhemostfavorable

regionwere84%,93.1%and89.6%,respectively.Moreover,intheadditionallyallowedregions,

nearly15.9%,6.9%and9.6%residuesofSARS-CoV–2spikeglycoproteinS2,viralNSP15

endoribonucleaseandmainprotease3CLprowerefound,respectively.ThetotalqualityG-factorof

themodeledviralreceptorssuggestedthatallofthemexhibitedfavourablestereo-chemical

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parametersandhence,the3Dstructuresofalltheseviralreceptorscorrespondtohigh-probability

conformationformoleculardocking.TheProCheckstereo-chemicalanalysisalsoshowednobad

contactsandscoresformain-chainorside-chainparameters.Thedetailedsecondarystructural

investigationoftheSARS-CoV–2spikeglycoproteinS2withPDBsumserverrevealedthat313(10.77%)

residueswereinstrands,2112(72.70%)residueswereinα-helices,78(2.68%)residueswereinβ-

turnsand402(13.83%)residueswereinotherconformations.Similarly,PDBsumsecondarystructure

ofNSP15endoribonucleaserevealedthat72(10.34%)residueswereinstrands,577(82.90%)

residueswereinα-helices,4(0.57%)residueswereinβ-turnsand43(6.17%)residueswereinother

conformations.Moreover,PDBsumsecondarystructureofmainprotease3CLprorevealedthat75

(12.66%)residueswereinstrands,457(77.19%)residueswereinα-helices,7(1.18%)residueswere

inβ-turnsand53(8.95%)residueswereinotherconformations(Figure2)..Thus,theaboveanalysis

suggeststhatthebackboneconformationandnon-bondedinteractionsofalltheselectedviral

receptorswerefoundtobewithinreasonablerangeandhencecanbeprocessedforfurtherdocking

analysis(Kumaretal.,2011).

3.3Activesiteanalysisofviralreceptors

ActivesiteanalysisofSARS-CoV–2spikeglycoprotein(S2),viralnuclease(NSP15endoribonuclease)

andprotease(MainProtease3CLpro)asconductedbyDoGSiteScorerindicatedthatthereare

variousactivepocketswithinthestudiedviralvirulencefactorswithdruggabilityrangingfrom0.12to

0.86(Table2)..ItwasfoundthatpocketsP_11(Drugscore:0.847),P_1(Drugscore:0.860)andP_0

(Drugscore:0.805)wereenergeticallyfavourableforperformingfurthermoleculardockingstudies

withtheviralreceptorsbeingspikeglycoprotein,NSP15endoribonucleaseandMainProtease3CLpro,

respectively.Whileconductingtheactivesiteanalysis,theDoGSiteScorertoolanalysedtheheavy

atomcoordinatesonthesurfaceofthe3Dstructureoftherespectiveviralreceptors.Dependingon

theseatomiccoordinates,ahypotheticalgridwasspannedbyoutrulingthechancesofanyspatial

overlapofthegridwiththeheavyatoms.Furthermore,thetoolengagesinapplyingaGaussianfilter

tothedefinedgrids,soastoidentifysphericalpocketsofbinding.Druggabilityscore(0–1)ofthe

selectedsphericalpocketsarededucedonthebasisoftheirsurfacearea,volume,enclosureand

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hydrophobicity.Asageneralrule,higherdruggabilityscoreisindicativeofamoredruggablepocket

(Volkameretal.,2012).

3.4Selectionofherbalsaspromisinganti-SARS-CoV–2candidates

Extensiveliteraturesurgecombinedwithamatrixbasedanalysiswasconductedforselectionof

plantshavingprobableutilityagainstSARS-CoV–2.Theparametersforselectingtheherbalsincluded-

a)ethnopharmacologicalimportanceoftheplant;b)priorpharmaco-therapeuticinvestigationsofthe

plant;andc)symptomaticreliefprovidingcapabilitiesoftheplant.Binary,weightageandfuzzyscore

analyseswereconductedforalltheplantssoastoscreenoutherbalsexhibitingprobableanti-SARS-

CoV–2activity.Plantsshowingpositiveassessmentformorethan03parameters,reportedinPubMed

searchengine(n=first20hits)wereselectedforfurtherinsilicoanalysis.Therationaleforselected

plantsalongwiththeirbinary,weightageandfuzzyscoreshasbeenexplainedinTable3.

Predominantphytocompoundsoftheselectedherbswerealsoidentifiedbyliteraturesurgeandthe

respectivechemicalstructureswereretrievedfromPubChemdatabase(Chakotiyaetal.,2014).

3.5Pharmacokineticdescriptorsofphytoligands

Druglikelinesscharacteristicsofthebioactivephytoligandswereassessedbyemployingastep-wise

filteringstrategy,whereinvariousphysiochemicalpropertiessuchaslogP,H-bondacceptor,H-bond

donor,molecularweight,acidicgroups,aromaticrings,numberofrotatablebondsandchains,

numberofhydrogenbondsandmolarrefractivitieswerepredictedsoastoevaluatethedruglike

behaviorofthephytoligand.AccordingtotheLipinski’srule,adruglikemoietyshouldhavealow

molecularweight(≤500D),logPvalue≤5,numberofhydrogenbondacceptors≤10,andnumberof

hydrogenbonddonors≤5.Abioactivedruggablemoleculeshouldensuetoatleast4ofthe5Lipinski

rules(Zhang&Wilkinson,2007).TheLipinskiscoresfortheselectedphytomoleculeswerefoundto

bewithinacceptablerangesaselucidatedinTable4.

3.6ADMEToxpredictionofphytoligands

TheresultsofadmetSARpredictionshowingtheprobabilityvaluesaresummarizedinTable5.The

phytoligandsviolatinganyoftheADMEToxdescriptors(amentoflavone,butenolide,malicacid,β-

myrcene,paeoniflorin)wereexcludedatthisstepitself.Restofthephytoligands(~12moieties)did

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notexhibitanymutagenicortoxicprofile.Basedonthepredictedprobabilityvalues,theselected

phytoligandswereknowntogetabsorbedefficientlybytheintestinalepitheliumasthevaluesfor

CaCo2permeabilityandintestinalabsorptionwerefoundtobewithinpermissiblerange(~absorption

value≥0.5).Suchpredictedabsorptionvaluessuggestthattheselectedphytoligandsmaygeteasily

transportedaftergettingabsorbedinthehumanbody.Theaqueoussolubilityoftheselected

phytoligandswasalsopredictedtobeacceptable(logS≥-4).Moreover,alltheselectedphytoligands

alsoexhibitedefficientbindingwiththeplasmaprotein,therebyensuringefficientdistributionofthe

probabledrugmoieties(Plasmaproteinbindingvalue≥0.5).Asfarasmetabolismisconcerned,

inhibitionpredictionsforcytochromeP450(CYP3A4)superfamilywereconducted.Noneofthe

selectedphytomoleculesexhibitedanyinhibitionofCYP3A4,thusensuringproperdrugmetabolism

andnoexcessivedrugaccumulationaswellastoxicity.MostoftheselecteddrugsalsoexhibitedP-

glycoproteinmodulationactivity(~P-glycoproteinactivationvalue≥0.5),therebymediatingactive

transportandmetabolismofthedrugmoieties.Othertoxicityandexcretioncharacteristicsalso

suggestedthattheselectedphytoligandsdidnotexhibitanysignsoforaltoxicity(~acuteoral

toxicityvalue≥1.0Kg/mol)andmutagenicity(Amesmutagenicityvalue≥0.5),thusmakingthe

respectivephytoligandsasprobableleadmolecules(Nishaetal.,2016).

3.7MolecularDockinganalysis

Dockingresultsoftheviralvirulencefactors,namely,spikeglycoprotein,NSP15endoribonuclease

andMainProtease3CLpro;andtheselectedphytoligands(~12phytomolecules)areshowninTable

6.ThesedockingbasedEvalueshavealsobeencomparedwiththatofthestandarddrug,i.e.,

hydroxychloroquine.HexbaseddockingresultsrevealedthattheE-valueofdockingofgamma-

glutamyl-S-allylcysteineandsalvianolicacidwithalltheselectedviralreceptors(viralmainprotease

3CLpro,spikeglycoproteinandNSP15endoribonuclease)wassignificantlybetterascomparedto

hydroxychloroquine.Severalotherphytoligandsalsoshowedcomparablebindingenergieswith

respecttoatleastoneoftheviralreceptors;however,noneoftheotherphyto-moietiesexhibited

holisticdockingabilities.Hence,itisobviousfromtheE-valuesthatgamma-glutamyl-S-allylcysteine

andsalvianolicacidbindspontaneouslyandirreversiblytoallthetestedviralreceptors,thereby

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blockingthespreadandreplicationofthenovelCOVID–19virus.Moreover,thebindingefficiencyof

gamma-glutamyl-S-allylcysteinehasbeenfoundtobeexceedinglybetterthanthatofsalvianolicacid.

Suchsignificantbindingaffinityofgamma-glutamyl-S-allylcysteineandsalvianolicacidpresumably

indicatestheprobablemechanismofactionofthesephytomoleculesasviralprotease,spike

glycoproteinandendoribonucleaseinhibitors.Suchefficientbindingenergyoftheseherbalmoieties

indicatesthemtobecomparablewiththatofhydroxychloroquinewhichhasbeenproposedasthe

cornerstoneforCOVID–19therapy.

Viralspikeglycoproteinisrequiredforinitiatingtheattachmentandentryofvirusintothehostcell.

Moreover,viralproteaseisfundamentalforcontinuingthevirallifecycleofSARS-CoV–2asitis

requiredbythevirustocatalyzethecleavageofviralpolyproteinprecursorswhichareultimately

necessaryforviralcapsidformationandenzymeproduction(Anandetal.,2003).Similarly,viral

endonucleasesarenecessaryforcatalyzingtheprocessingofviralRNAsandhencearerequiredfor

enduringtheprocessofviralreplication(Balkrishnaetal.,2020).Henceforth,gamma-glutamyl-S-

allylcysteineandsalvianolicacidcontingentlyinhibitstheentryofSARS-CoV–2andalsoinactivates

theviralproteaseaswellasendoribonuclease,therebyinhibitingtheprocessofviralcapsidformation

andreplicationaltogether.Earlierstudieshavealsoindicatedthatgammaglutamylcysteineester

derivativescouldinhibittheHIV–1genetranscription,wherein,itprobablyrestrainedtheoxygenfree

radical-mediatedactivationofthenuclearfactor-kappaB(NF-κB).Moreover,thismoietyhasalonger

plasmalife,bioavailabilityandmembranepermeability,therebymakingitasuitabledrugcandidate

withsustainedantiviralaction(Kubotaetal.,1998).

3.8PhytoligandandViralreceptorbindingposedepictions

Thebestdockedconformationswithlowestdockingenergy,i.e.,gamma-glutamyl-S-allylcysteineand

salvianolicacidwereselectedforfurtherMDsimulationsusingPoseViewforcreatingposedepictions

ofselectedligand-receptorbinding.Uponassessingthebindingposeandelectrostaticbridging

interactions,itwasfoundthatonlygamma-glutamyl-S-allylcysteine(Herbalsource:Alliumsativum)

wasabletofitintotheactivebindingpocketsoftheviralreceptors,whereas,salvianolicacidcould

notestablishanirreversibleandspontaneousbondwiththeviralreceptors.Theorientationalbinding

14

ofgamma-glutamyl-S-allylcysteineandtheviralreceptorsshowingtheposeviewandresidue

interactionshavebeendepictedinFigure3.Itwasobservedthattheamidegroupofgamma-

glutamyl-S-allylcysteineformedahydrogenbondwiththeamideresidueofglutamineaminoacid

(1071stposition)foundintheviralspikeglycoprotein.Chemicalbridgingofgamma-glutamyl-S-

allylcysteineandglutamicacidresiduesofviralendoribonucleasepresentasimilarcasewhere

glutamicacidresidues(45thposition)wereinvariablyboundandneutralized,therebypossibly

neutralizingtheCOVID–19virus.Inearlierstudiesithasbeenfoundthatmutationoranychangein

theglutamineorglutamicacidresiduesofEbolavirusspikeglycoproteincausesviralneutralization

(Reynard&Volchkov,2015).Similarly,thehydroxylgroupofgamma-glutamyl-S-allylcysteineformed

ahydrogenbondwithcarbonylgroupofprolineaminoacid(108thposition)ofviralmainprotease.

Previousstudieshavealsosuggestedthattheprolineaminoacidresiduesfoundintheconserved

domainsofHIVviralinfectivityfactor(Vif)aretherapeutictargetsforneutralizingthehuman

immunodeficiencyvirus(Ralphetal.,2020).Inconclusion,thebindinginteractionsofgamma-

glutamyl-S-allylcysteinewithviralmainprotease,spikeglycoproteinandendoribonucleasesuggestit

tobeapromisingdrugcandidateformitigatingnovelCoronavirusinfection.

4.ConclusionThenovelCoronavirusinfectionaccountsforinnumerabledeathsworldwide,andthereisyetno

approvedprophylacticvaccineorspecifictreatmentavailable.Basedonevidencefromlaboratory,

animalandclinicalstudies,Hydroxychloroquineisoneofthetreatmentoptionsselectedin

‘Solidarity’-aninternationalclinicaltrialtohelpfindaneffectivetreatmentforCOVID–19,launchedby

theWorldHealthOrganizationandpartners.However,therepurposeddrug,hydroxychloroquinemay

causeadversedrugreactionsandcontraindicationsincludingcardiomyopathy,fulminanthepatic

failure,vertigoandotherallergicreactions.Undersuchcircumstances,thereisanurgentneedfor

screeningnovelnaturalleadsthatexhibitspecificantiviralactivitiesagainstSARS-CoV–2.Thepresent

studysuggestedthatphytoligandsderivedfrommedicinalherbsexhibitedpotentialbinding

propertiestowardmajorSARS-CoV–2virulencefactors.Selectedphytomoleculeswerefurther

screenedonthebasisofacceptablepharmacokineticanddrug-likeproperties,therebymakingthem

15

safelyexploitableforCoronavirusmitigationsystem.Thecurrentstudyshowedthatgamma-glutamyl-

S-allylcysteine(GGA)specificallyexhibitedthemostsignificantbindingenergyanddockingpose

towardthemajorviralvirulencefactors(EvalueGGA+spikeglycoprotein=–578.57Kcal/mol;EvalueGGA

+viralmainprotease=–493.53Kcal/mol;EvalueGGA+endoribonuclease=–825.00Kcal/mol)incomparison

withtheknownchemicalmoietyhydroxychloroquine(EvalueHCQ+spikeglycoprotein=–207.47Kcal/mol;

EvalueHCQ+viralmainprotease=–235.48Kcal/mol;EvalueHCQ+endoribonuclease=–213.54Kcal/mol).

Hence,thecurrentstudyprovidesimplicationtowardthepossibleusageofgamma-glutamyl-S-

allylcysteine(Herbalsource:Alliumsativum)asanovelandprospectivedrugcandidate.This

phytomoleculeisalsofoundinotherspeciesoftheAlliumgenus(A.cepaandA.schoenoprasum)..In

viewofthecurrentviralpandemicanddearthofeffectivetherapy,furtherstudiesshouldbeurgently

undertakensoastoexplorethetherapeuticpotentialofgamma-glutamyl-S-allylcysteineagainst

SARS-CoV–2.

AbbreviationsACE2:Angiotensinconvertingenzyme2;ADMETox:Adsorption,Distribution,Metabolism,Toxicity;AP–

1:Activatorprotein1;ATF–2:Activatingtranscriptionfactor2;CoV:Coronavirus;COVID:Coronavirus

disease;DARS:DecoysasReferenceState;DoG:DifferenceofGaussian;FFT:FastFourierTransform;

GGA:Gamma-glutamyl-S-allylcysteine;GPCR:G-proteincoupledreceptor;HCQ:Hydroxychloroquine;

IRF:Interferonregulatoryfactor;MERS:MiddleEastRespiratorySyndrome;NF-Kβ:NuclearFactor

kappa-light-chain(β);NSP:Nonstructuralprotein;O.E.C.D.:OrganisationforEconomicCo-operation

andDevelopment;PDB:ProteinDataBank;QSAR:QuantitativeStructureActivityRelationship;RCSB:

RoyalCollaborativeStructuralBiology;SARS:SevereAcuteRespiratorySyndrome;TPSA:Totalpolar

surfacearea;VIF:Viralinfectivityfactor

DeclarationsAuthorContributions

ABandRKSconceivedthepresentedresearch.PT,SSandSNDanalyzedtheinformation,generated

theartwork,andco-wrotethemanuscript.AB,NSandRKSinvestigatedandsupervisedthefindingsof

thework.RKSprovidedcriticalrevisionofthisarticle,andapprovedthemanuscriptforsubmission.

16

Allauthorsagreedwiththefinalversionofthismanuscript.

Funding

Noexternalfundinghasbeenreceived.

EthicsStatement

Theauthorsconfirmthattheethicalpoliciesofthejournal,asnotedonthejournal’sauthorguidelines

page,havebeenadheredto.Noethicalapprovalwasrequiredasthisisaninsilicostudy.

ConflictofInterest

Theauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancial

relationshipsthatcouldbeconstruedasapotentialconflictofinterest.

Acknowledgments

TheauthorsaregratefultoSwamiRamdevJiforinstitutionalresearchfacilitiesandsupports.Authors

gratefullyacknowledgetheeffortsofcolleaguesofPatanjaliResearchInstitutefortheirhelpindata

collectionandprocessing.WearealsothankfultoMr.GaganKumarandMr.LalitMohanfortheirswift

administrativesupportsandencouragements.

DataAvailabilityStatement

Alldatageneratedoranalysedduringthisstudyareincludedinthispublishedarticle.

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TablesTable 1. Selection of COVID-19 virulence factors on the basis of relevance score as assessed bykeywordhitsscoringmatrix

21

Parameter Rationaleofselection TotalNo.ofHits(N)

HitsScreened(n)

RelevantHits(r)

PercentageRelevance†

ViralSpikeGlycoproteininhibitor

Enveloped viruses enter cells by viralglycoprotein-mediated binding (Viral spikes –S proteins) to host cells and subsequentfusionof virusandhost cellmembranes (Liuetal.,2020).

20 20 18 90

ViralEndoribonucleaseinhibitor

Endoribonuclease catalyses the processingand degradation of both cellular and viralRNAs, thus determining the amount andfunctionality of specific RNA molecules in acell at any given time. It degrades the hostmRNA,whilecleavestheprecursorviralRNAstoproduceactivegeneticmaterial(Balkrishnaetal.,2020).

103 20 13 65

ViralProteaseinhibitor

Viral proteasesareenzymesencodedby thegenetic material of viral pathogens so as tocatalyse the cleavage of specific peptidebonds in viral polyprotein precursors or incellular proteins. The protease cleaves theprecursor viral polyprotein to producefunctional proteins and enzymes (Jo et al.,2020).

1516 20 12 60

Anti-bronchitisHerb

Coronavirus causes respiratory illnesses.Hence, herbs providing symptomatic reliefagainstrespiratorysymptomsmightprovetobebeneficial(Carlosetal.,2020).

551 20 9 45

Anti-gastroenteritisHerb

Coronavirusalsocausesgastroenteritis.Thus,herbs providing symptomatic relief againstgastrointestinal ailments might prove to bebeneficial(Chenetal.,2020).

52 20 8 40

Interferonregulatoryherb

Interferons (IFNs) are cytokines which areused for communication between cells totriggertheprotectivedefenseoftheimmunesystem that help eradicate pathogens,Coronavirus in this case (Balkrishna et al.,2020).

952 20 6 30

Duetotechnicallimitations,table2isonlyavailableasadownloadinthesupplemental

22

filessection.

Table3.SelectedHerbalmoietiesshowingprobableantiviralutilityasassessedbyemployingextensiveliteraturesurge.

PlantSource§ Predominantphytocompound

Activityexplored† BinaryScore

WeightageScore

FuzzyScore*

ProbableantiviralutilityS2 NSP

153CLpro

AlismacanaliculatumA.Braun&C.D.Bouché

AlisolA24-Acetate + − − 2 1.16 0.46 Anti-influenzaactivityobserved as theherbal moietyinactivates thehemagglutininspikereceptor.

AlliumcepaL. Allicin + + + 3 1.66 0.66 Hinders virusattachment tohost cell, altertranscriptionandtranslation ofviral genome inhost cell andalso affect viralassembly.

AlliumsativumL. Gamma-Glutamyl-S-allylcysteine

+ + + 6 2.49 1 Actsasproteaseinhibitormainly.

ArtemisiacapillarisThunb.

Beta-caryophyllene + − + 5 1.91 0.76 Symptomaticalleviation incaseofhepatitisvirusinfection.

ArtemisiacaruifoliaBuch.-Ham.exRoxb.

Caruilignan + − + 3 1.25 0.5 Anti-influenzaand anti-herpessimplex virusactivity; alsoinhibits HIV-1protease.

AsparagusracemosusWilld.

Isoasparagine − − − 3 0.40 0.3 Symptomaticalleviation incase of herpesvirusinfection.

BerberisaristataDC. Berberine + − + 5 1.91 0.76 Inhibitsenterovirus 71entry andreplication bydownregulatingthe MEK/ERKsignalingpathway andautophagy.

BoswelliaserrataRoxb. 11-keto-beta-boswellicacid

+ − + 3 1.66 0.66 InhibitsChikungunyaand Vesicularstomatitis virusinfections byblocking theirentry.

Camelliasinensis(L.) Quercetin + − + 5 1.91 0.76 Suppressed

23

Camelliasinensis(L.)Kuntze

Quercetin + − + 5 1.91 0.76 SuppressedHepatitisCvirusentry, and alsoinhibited viralRNAreplication.

ChlorophytumborivilianumSantapau&R.R.Fern.

Neotigogenin − − − 2 0.40 0.3 Cytokinemodulatingpotential.

CurcumalongaL. Curcumin + − + 5 1.91 0.76 Inhibits entry ofChikungunyaand Vesicularstomatitisvirus.

EpimediumflavumStearn

Wushanicariin + − − 1 1 0.4 Induced thesecretionoftypeI IFN and pro-inflammatorycytokines.

GingkobilobaL. Amentoflavone + − + 4 1.66 0.66 Inhibits viralprotease,specifically incase of HIVinfection.

HouttuyniacordataThunb.

β-myrcene + − + 5 1.91 0.76 Inactivation of3C-likeproteinase ofmurineCoronavirus anddenguevirus.

MelissaofficinalisL. Citronellal + + − 5 1.49 0.59 InhibitionofHIV-1protease.

OcimumtenuiflorumL. Carvacrol − − + 3 0.75 0.35 Inactivation ofviral protease incase of HIVinfection.

PaeonialactifloraPall. Paeoniflorin + − − 3 1.41 0.56 Inhibits viralentry in case ofInfluenza virusinfection.

PhyllanthusamarusSchumach.&Thonn.

Gallotannin − + + 5 1.49 0.59 Halts theprocess of viralreplication incase of Herpessimplex virusinfection.

RheumrhabarbarumL. Malicacid + − + 4 1.91 0.76 Inhibits viralentrybyceasingthe endosomalfusion incaseofinfluenzavirus.

SalviamiltiorrhizaBunge

Salvianolicacid − + + 5 1.49 0.59 InhibitionofHIV-1 integrase andprotease.

Taxillussutchuenensisvar.duclouxii(Lecomte)H.S.Kiu

Butenolide + − + 2 1.5 0.60 Inhibition ofHepatitis C viralNS3 serineprotease andceasing viral

entry.Tinosporacordifolia(Willd.)Hook.f.&Thomson

Tinosporaside + + + 5 2.49 1 Immunomodulatory activity;Anti-HIV activitywherein it acts

24

wherein it actsas viralribonucleaseinhibitor.

Withaniasomnifera(L.)Dunal

Withanolide + + + 3 1.41 0.56 Disruptsinteractionsbetween viral S-protein receptorbinding domainand Host ACE2receptor.

ZingiberofficinaleRoscoe

6-Gingerol − + + 5 1.49 0.59 InhibitsHepatitisCvirusprotease.

§Plants are selectedon thebasis of extensive literature surge, specifically focusingon their ethno-medicinalattributes,symptomaticreliefprovisionabilitiesanddirect/indirectantiviralactivity,ifany.†Symbolsof+and–denotethepresenceandabsenceofviralvirulencefactorinhibitorypropertiesinthe given plant, as deduced on the basis of keyword searchmatrix analysis using PubMed searchengine.*Fuzzy score μS= (S-minS)/ (maxS-minS), wherein shaded cells represent the ligands selected forfurtherstudywithafuzzyscore>0.5.

Table 4. Physicochemical properties of phytoligands in comparison with the standardchemotherapeuticagent.

Ligand/StandardPhysicochemicalProperties

Mol.Wt.(≤500D)

LogP(≤5)† H-BondDonor(≤5)

H-DonorAcceptor(≤10)

Lipinskiviolations(ifany)*

Allicin 162.02 0.237 0 1 0Amentoflavone 538.09 2.030 6 10 1

Berberine 336.12 2.473 0 4 0Beta-caryophyllene 204.19 6.044 0 0 111-keto-beta-boswellicacid

470.34 8.131 2 4 1

Butenolide 84.02 0.308 0 2 0Citronellal 154.14 3.591 0 1 0Curcumin 368.13 1.945 2 6 0Gallotannin 1700 9.537 25 46 4

Gamma-Glutamyl-S-allylcysteine

290.09 -2.68 4 7 0

6-Gingerol 294.18 2.437 2 4 0Malicacid 134.02 -1.474 3 5 0β-myrcene 136.13 4.170 0 0 0Paeoniflorin 480.16 -0.464 5 11 1Quercetin 302.04 1.834 5 7 0Salvianolicacid 494.12 2.898 7 10 1Tinosporaside 492.20 0.54 4 10 0Withanolide 470.27 3.263 2 6 0Hydroxychloroquine 335.88 4.00 4 2 0

†Logarithmofcompoundpartitioncoefficientbetweenn-octanolandwater.*Shadedcellindicatesphytoligandwithmorethan1Lipinskiviolationsandhenceiseliminatedatthis

stageitself.

Table5.ADMEToxvaluesofphytoligandsincomparisonwiththestandardchemotherapeuticagent.

25

Ligand/Standard

Absorption Distribution Metabolism Excretion

Caco-2permeability(value≥0.5)

Humanintestinalabsorption(value≥0.5)

PlasmaProteinbinding(value≥0.5)

Watersolubility(logS≥-4)

P-glyco-proteinactivator(value≥0.5)

CYP3A4inhibition(value≥0.5)

Acuteoraltoxicity(Kg/mol)(value≥1.0)

Allicin 0.58(+) 0.91(+) 0.50(+) -0.89(+) 0.98(+) 0.92(-) 1.935(-)Amentoflavone 0.87(+) 0.98(+) 1.11(+) -3.36(+) 0.44(-) 0.61(-) 1.822(-)

Berberine 0.94(+) 0.77(+) 0.83(+) -2.97(+) 0.68(+) 0.58(-) 1.545(-)

Beta-caryophyllene

0.86(+) 0.98(+) 0.83(+) -4.68(+) 0.89(+) 0.86(-) 2.366(-)

11-keto-beta-boswellicacid

0.54(+) 0.99(+) 1.05(+) -3.45(+) 0.63(+) 0.79(-) 2.834(-)

Butenolide 0.76(+) 0.96(+) 0.096(-) 0.23(+) 0.98(+) 0.98(-) 1.976(-)

Citronellal 0.92(+) 0.97(+) 0.70(+) -2.44(+) 0.98(+) 0.96(-) 2.307(-)

Curcumin 0.76(+) 0.97(+) 0.83(+) -3.36(+) 0.59(+) 0.53(-) 1.992(-)

Gamma-Glutamyl-S-allylcysteine

0.92(+) 0.63(+) 0.50(+) -1.68(+) 0.93(+) 0.74(-) 1.648(-)

6-Gingerol 0.59(+) 0.99(+) 0.85(+) -3.23(+) 0.89(+) 0.59(-) 2.290(-)

Malicacid 0.95(+) 0.77(+) 0.23(-) 0.27(+) 0.98(+) 0.90(-) 0.844(+)

β-myrcene 0.77(+) 0.96(+) 0.43(-) -3.44(+) 0.98(+) 0.66(-) 1.660(-)

Paeoniflorin 0.82(+) 0.41(-) 0.67(+) -2.97(+) 0.65(+) 0.85(-) 3.502(-)

Quercetin 0.64(+) 0.98(+) 1.17(+) -2.99(+) 0.91(+) 0.69(-) 2.559(-)

Salvianolicacid 0.93(+) 0.96(+) 1.03(+) -3.20(+) 0.65(+) 0.83(-) 2.069(-)

Tinosporaside 0.84(+) 0.83(+) 0.50(+) -3.65(+) 0.54(+) 0.75(-) 3.236(-)

Withanolide 0.62(+) 0.97(+) 1.18(+) -4.00(+) 0.51(+) 0.85(-) 3.660(-)

Hydroxy-chloroquine

0.66(+) 0.99(+) 0.86(+) -4.00(+) 0.84(+) 0.83(-) 2.684(-)

*Denoted ‘+’ or ‘-’ sign relates to the presence or absence of a predicted activity, respectively.Shadedcellsindicatethedescriptorsviolatingthestandardvalues,therebyexcludingtherespectivephytoligand(s)fromfurtherstudies.

Duetotechnicallimitations,table6isonlyavailableasadownloadinthesupplemental

filessection.

Figures

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Figure1

MolecularPathogenesis&ClinicalmanifestationsofNovelCoronavirus(SARS-CoV-2).SARS-

CoV-2infectslungparenchymalcellsanditsentryisfacilitatedbybindingofviralenvelope

glycoprotein(S2)withhostACE2receptor.Uponenteringthehostbronchialcells,thevirus

elicitsanimmunologicalresponseasmediatedbyhostgenes,namely,TLRs-dependentIFN

inductionpathways[IRF-3/7(1)&NF-Kβ(2)]andmyeloid-differentiationprimaryresponse

88pathways[ATF-2(3)&AP-1(4)].Subsequently,inflammatorycytokines(IL-6,IL-12and

IL-8),interferongammaandreactiveoxygenspecies(ROS)areproduced.These

inflammatoryandoxidationmediatorsactivateapathogenesiscascade,leadingto

inflammation,alveolarmucusformationanddecreaseinpartialpressureofoxygenin

bronchioles.SimultaneouslySARS-CoV-2getsholdofhostmetabolicmachineryandinitiates

itsreplicationandtranslation,asmediatedbytheprimaryviralvirulencefactors(RNA

27

dependentRNApolymerase/endoribonuclease,andviralmainprotease).Asaconsequence,

viralloadinbloodincreases(viraemia),ultimatelyleadingtoseriousclinicalmanifestations

includingfever,alveolaredema,hypoxemia,dyspnoea,coughandmultipleorganfailure.

28

Figure2

RamachandranplotofthestructuremodelsofSARS-CoV-2receptors-(A)Spike

glycoprotein,(B)NSP15endoribonuclease,and(C)Mainprotease3CLpro.Themost

favouredregionsarecolouredinredandmarkedasA,B,andL.Theadditionallyallowed

regionsarecolouredinyellowandmarkedasa,b,l,andp.Allnon-glycineandproline

residuesareshownasfilledblacksquares,whereasglycineresidues(non-end)areshown

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asfilledblacktriangles.Disallowedresidues,ifanyarerepresentedbywhitecolour.

Abbreviations:Asn:asparagine;Asp:aspartate;Gly:glycine;Pro:proline.

Figure3

Bindingposedepictionsofgamma-glutamyl-S-allylcysteineasprobabledrugcandidates

targetingSARS-CoV-2receptorsAmidegroupofgamma-glutamyl-S-allylcysteine(GGA)

formedahydrogenbondwiththeamideresidueofglutamineaminoacid(1071stposition)

oftheviralspikeglycoprotein.Glutamicacidresidues(45thposition)ofviral

endoribonucleaseshowedirreversibleinteractionwithGGA.Additionally,thehydroxylgroup

ofGGAformedahydrogenbondwithcarbonylgroupofprolineaminoacid(108thposition)

ofviralmainprotease3CLpro.

SupplementaryFilesThisisalistofsupplementaryfilesassociatedwiththispreprint.Clicktodownload.

GraphicalAbstract.jpgTable2.docxTable6.docx

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