identification of novel drug targets and lead compounds in

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Research Article

DOI: 10.21767/2470-6973.100003

Chemical informaticsISSN 2470-6973

1© Under License of Creative Commons Attribution 3.0 License | This article is available in: www.cheminformatics.imedpub.com/archive.php

Varun Khanna1, Ashutosh Kumar1 andRishi Shanker1

Institute of Life Sciences, AhmedabadUniversity,Navrangpura,UniversityRoad,Ahmedabad,Gujarat,India

Corresponding author: VarunKhanna

[email protected]

Institute of Life Sciences, AhmedabadUniversity, Navrangpura, University Road,Ahmedabad,Gujarat,India.

Tel: +91 79 26302414-18 Fax: +91 79 26302419

Citation: Khanna,KumarA,ShankerR.IdentificationofnoveldrugtargetsandleadcompoundsinAnthraxandPneumoniacausingpathogensusinganInSilicoapproach.ChemInform.2015,1:1.

Identification of novel drug targets and lead compounds in Anthrax and Pneumonia

causing pathogens using an In Silico approach

IntroductionThe arrival of post-genomic era has ushered the possibility ofhigh-throughputscreeningofcompoundlibrariesagainstallthepotentialtargetsofapathogen.However,apersistentbottleneckinstructurebaseddrugdiscoveryistheavailabilityofdruggabletarget which play a vital role in parasite and is sufficientlydifferent from human counterpart. Experimental validation ofidentifiedpotentialdrugtargetsisanadditionalchallenge,asitisimpracticalforgenomewidescales.Therefore,severalin silico approachessuchasdiscoveringkeystepsinmetabolicpathways,chemogenomics, subtractive and comparative genomics havebeenproposedtoidentifyandprioritizedrugandvaccinetargetsfordifferentpathogens[1-4].

Followingisabriefsummaryofstudiesondrugtargetidentificationand validation. In oneof the studies a combinationof in silico

andexperimentalapproachesidentified52proteinsasprobablevaccine candidates for highly invasive Indian Streptococous pyogenesserotypes[5].Thisstudyalsorevealedthatthehighlyinvasive group A Streptococous M49 had metabolically moreactivemembraneassociatedproteinmachinerythanlessinvasiveM1 strain. Caffrey et al. [6] compared the putative proteomeof Schistosoma mansoni with the two model organismsCaenorhabditis elegans and Drosophila melanogaster usingGenlightsoftware.Theauthorswereabletoidentify35druggableproteins in S. mansoni, 18 of which were homologous to theproteinswith3Dstructuresincludingco-crystallizedligands.The

in silico and in vitro screening for potential vaccine candidatesandvirulencerelatedgenesintheB. anthracisvirulenceplasmid

pXO1haspreviouslybeeninvestigatedin[7].Theknown143openreading frames (ORFs) in pXO1were subjected to comparative

AbstractIncreased resistance to antibiotics and delay in treatment significantly reducethechancesofsurvivalforanthraxandpneumoniapatients.Inthepresentstudycomparativeproteomeanalysis and virtual screening approacheswereused todiscover novel therapeutic drug targets and antibacterial lead compounds forBacillus anthracis andBacillus cereus strainsresponsibleforcausinganthraxandpneumonia,respectively.Theemployedsystematicworkflowidentifiedatotalof803proteins,withinthegenomesoftheaforesaidpathogens,withnosignificantsequence similarity to human proteins. Out of 803 non homologous proteins,10were found to be essential for the survival of pathogens. Further basedonnumberofcriteriaincludingessentiality,druggabilityandavailabilityofstructuralinformation thioredoxin reductase (TrxR) was shortlisted as the potential drugtarget.In silicodockingstudieswiththreeopensourcedockingprogramsidentifiedfour potential inhibitors of bacterial TrxR.. In conclusion this study resulted inidentificationofanoveldrugtarget(Thioredoxinreductase)forBacillus anthracis andBacillus cereus previouslynotknownofthesepathogenicmicroorganisms)andpotentialinhibitorsofTrxRthatcouldfacilitateinselectionofdrugleadsforentryintodrugdesignandproductionpipelines..Inaddition,themethodologyusedcanbeappliedfortheidentificationofnewdrugtargetsanddrugleadsincaseofotherpathogenicmicroorganisms.

Keywords: Bacillus, Anthrax, Pneumonia, Drug target identification, Moleculardocking,Thioredoxinreductaseinhibitors

Received: June30,2015; Accepted: August19,2015;Published: August24,2015

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genomicanalysiswithrelatedgenomesofBacillus subtilis,Bacillus halodurans, Bacillus cereusand thepBtoxisplasmidofBacillus thuringiensis var. Israeliensis. As a consequence, authors wereabletoannotatemorethan60%ORFscomparedto30%knownearlier.Subsequently,nineORFsweresuccessfullyexpressedasfull lengthpeptidesand threewere found tobeantigenicwithimmunogenicpotential[7].Crowtheret al.[8]revieweddifferentstrategiestoprioritizepathogenproteinsbasedonthepropertieswhichareconsidereddesirablefordrugtargets.Theauthorsalsosuggestednoveldrugtargetsforseventropicaldiseasepathogensincluding Mycobacterium tuberculosis, Plasmodium falciparum andothers.

Inthecurrentstudy,in silicoapproachwasusedtodiscovernoveldrugtargetsinanthraxandpneumoniacausingstrainsandsub-strainsofBacillus.Thediscoveredtargetswerefurthersubjectedto virtual screening in order to identify novel lead compoundsinhibitingthosetargets.WeusedthreestrainsofBacillus (viz. B. anthracis, B. cereusandB. toyonensis)whichareGram-positive,endospore-forming, rod-shaped bacteria and have a similargeneticbackground.B. anthracis(thecausativeagentofanthrax)existintheenvironmentasasporeandcanremainviableinsoilfordecades[9].Sporesingestedbygrazingherbivoresgerminatewithintheanimalandproducethevirulentvegetativeformsthatreplicateand eventually kill the host. Products (e.g., meat or hides) frominfectedanimalsserveasareservoirforhumandisease[9].Recently,there has been an increase in the number of cases of “injectionanthrax" in European countries, including Denmark, France, andtheUnitedKingdom(aformofdiseasethataffectsheroinusersandis causedbyB. anthracis) [10]. B. cereuson theotherhand isanopportunistic,non-specifichumanpathogenresponsibleforcausingpneumonia[11,12],foodpoisoning[13]nosocomialinfectionsandcontaminationinhospitals[14].Itfrequentlypollutesdairiesduetotoxinsynthesisandformationofheat−resistantspores,whichsurvivepasteurization[15].Itisalsoconsideredasoneofthemostsevereorganismthataffecttheeye(mostoftenaftertrauma)resultinginblindness[16].B. toyonensis isanon-pathogenicstrainwhichwasfirstisolatedinJapanin1966.Itisbeingusedasaprobioticinanimalfeedsince1975[17].

Theexistingdrugsandvaccinescurrentlyavailableforthetreatmentof anthrax disease and pneumonia are effective only when thetreatmentisinitiatedsoonafterearlydiagnosis.However,delayintreatment significantly reduces the chances of survival. Anotherproblemwithmostoftheexistingantimicrobialdrugsistheirinabilitytoactonthelatentformsofbacteria.Thisstronglysuggeststheneedfornewtherapeutic targetsandnoveldrugorvaccinecandidatesthatofferbetterprotection.

Therefore, in thepresentstudy,wepredictedandvalidatednoveldrugtargetsusingin silicoapproaches.Additionally,wescreenedalibraryofcompoundsfromZINCdatabasetopredictleadmoleculesofhighbindingaffinityandselectivityfortheidentifiedtargets.Theapproachused,canbeappliedfortheidentificationofnoveldrugtargetsanddrugleadsincaseofotherpathogenicmicroorganisms.

Material and methodsData assembly, clustering and identification of orthologous proteinsAsystematicworkflowinvolvingvariousbioinformaticsmethods,tools and databases for identification of novel drug targets in

pathogenic strains ofB. anthracis and B. cereus was designed(Figure 1). The complete proteomes for B. anthracis (strainA0248 andH9401), B. cereus (strain B4264 and 03BB102) andB. toyonensis were downloaded fromUniprot database (www.uniprot.org).5291and5788proteinsequencesfromstrainA0248and H9401 respectively were combined in one file. Similarly,5598and5384proteinsequencesfromB. cereusstrain03BB102andB4264 respectivelyweremerged inotherfile.Using singlelinkage clustering algorithm implemented in BLASTClust toolavailablefromNCBI-BLASTpackage[18],datasetswereclusteredusing 90% sequence coverage and 95% sequence identity. Theprogrambeginswithpairwisematchesandplacesasequenceina cluster if the sequencematches at least onemember in thecluster.ClusteringwasperformedinordertoremoveredundancyandgetauniquesetofsequencesfromB. anthracisandB. cereus strains.Foridentificationoforthologsclusteredsequenceswerethen subjected to BLASTP analysis with the e-value inclusionthresholdsettoe-10.AsubsequentBLASTsearchagainstthenon-pathogenic strain (B. toyonensis) elicited the set of sequencesthatwerepresentinpathogenicstrainsbutwereabsentinnon-pathogenicstrain.

Identification of non-homologous and essential proteinsProteinsequencesexclusivetopathogenicstrainswerecomparedwith the non-redundant database of human proteins usingBLASTP to reveal non homologous set of proteins. Sequenceswith no significant sequence similarity to human proteins(non homologous set) were further filtered by searching forhomologoussequencesinthedatabaseofessentialgenes(DEG)[19]. The DEG database search was performed to identify thecritical genes necessary for the survival of pathogens. Thehitswithe-valuelessthane-10andwiththeminimumbitscoreof100havingsamenameandfunctionwereconsideredashomologousto essential proteins. The e-value used in this study was asdescribedbyGoshet al.[20].

Identification of potential drug targetsIn order to identify potential drug targets, identified essentialproteinswerealignedtothetargetsofallFDA-approved,smallmolecule, nutraceutical and biotech drugs in DrugBank (DB)database [21]. The alignments with e-value less than (1E-25)werediscardedexceptfortwoproteins(UniProtID: I0D343andI0D5Y2)becauseoftheavailabilityoftheircounterpartsinotherpathogenicorganismspresentinDBdatabase.Tofurthernarrowdown our search for the potential drug targets we searchedfor the availability of X-ray structures of proteins bound withanatural ligandoradrug in theProteinDataBank (PDB) [22].Structural information about the protein target could enhancethedrugdiscoverybyfacilitatingthestructurebaseddrugdesignincludingdockingandhighthroughputvirtualscreening.

Virtual screeningThevirtualscreeningworkflowhasbeendescribed inFigure 2. Wenarroweddowntoasingletargetcalledthioredoxinreductase(TrxR) for the reasons discussed under results and discussionsection. Initially ten inhibitors of M. tuberculosis thioredoxinreductase (MtTrxR) with known IC50 values were taken from

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theChEMBLdatabase[23].Subsequently,a librarywascreatedby exhaustively searching ~12 million compounds of thepurchasable clean subset from theZINCdatabase.This yielded167 compounds with the similarity score > 0.6. Similarity wasmeasured using Tanimoto coefficient on the basis of extendedconnectivityfingerprints implemented inSciTegicPipelinePilot.Finally, virtual screening campaign was launched to identifypotential small molecule inhibitors of B. anthracis thioredoxinreductase(BaTrxR)thattargettheprotein-proteininteractionsite(PPI)forthesubstratethioredoxin(Trx).

Structural comparison of Bacillus anthracis TrxR (BaTrxR)Thebacterialthioredoxinreductaseismadeupoftwodomains:theNADPH-bindingdomainandFAD-bindingdomain(Figure 3).There is ample evidence that during the enzyme catalysis theoxidisedconformation(FO)convertstothereducedconformation

(FR)by the rotationof theNADPHdomainabout its axisby67degrees.Formoredetailsontheconformationalchangesduringbinding of Trx in bacterial thioredoxin reductase readers couldrefer [24]. Most of the structures of TrxR in PDB disclose theFO conformation including the PDB structure of BaTrxR (4FK1)and MtTrxR (28A7). 1F6M is the only X-ray structure from E. coli that shows reduced conformation therefore, the reducedconformationofBaTrxRwasmodelledusing thecoordinatesof1F6M.

Additionally, the most prominent interactions reported duringbindingofEcTrxtoEcTrxRareobservedbetweentheEcTrxloop(Try 70 - Ile75) and complementary pocket on EcTrxR surface.Arg73sidechainoftheEcTrxpenetratesdeepinsidethepocketand forms hydrogen bonds with the backbone carbonyl ofAla237andArg130ofthereceptor(Figure 4).Otherimportantinteractions are seen between Asp 139 side chain of EcTrxR

Figure 1 Overallschematicsofthemethodologyadoptedforidentificationofnoveldrugtargetsanddrugleads.

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and the backbone amide of EcTrx Ile75. The importance ofArg73hasbeendemonstrated inapreviousstudywheretheloss of activity was observedwhen Argwasmutated to GlyandAsp[25].DuetohighstructuralsimilaritybetweenEcTrxRandBaTrxRasimilarbindingmodecanbeexpectedofBaTrxtoBaTrxRcharacterizedbyhydrogenbondingtoAla223andAsp137.Therefore, thebackbonecarbonylgroupofAla223andthecarboxylateofAsp137wereselectedastheprimarytargetpointsforpotentialinhibitors.

Preparation of the proteinThe comparison of FO and FR conformations of BaTrxR andEcTrxR shows that FAD domain simply rotates away whilebinding Trx, without affecting Trx binding. This allowedcreatingtheFRmodelofBaTrxRbysimply removingtheFADdomain. Subsequently, Dock Prep tool inUCSF Chimera [26]wasusedtocompletethereceptorpreparation.

Preparation of the ligandsCompoundsobtainedaftersearchingtheZINCdatabasewereprepared using Structure PrOtonation REcognition System(SPORES) [27]. For this manually added hydrogen atoms intheligandswerefirstremovedandthenautomaticallyaddedby the software. SPORES generates all possible protonationstates using rule a rule based combinatorial method. Next,all combinatorial possible tautomers of the inputmoleculeswere generated and stored in a multi-mol2 file. Finally thecompoundswereenergyminimizedusingMMFF94forcefieldasimplementedinopenbabelversion2.3.2[28].

Docking procedure Dockingwascarriedoutusingthreedifferentdockingprogramsviz.UCSFDOCKversion6.7 (DOCK) [29],AutodockVina (VINA)

[30] and Potein-Ligand ANT System (PLANTS) version 1.2 [31].The compoundswhichwere found to be common in all threedockingresultswereconsideredasfinalpredication.

DOCK setupAfter the initial preparation of the receptor, DOCK specific stepsrequired for preparing the binding site prior to docking wereimplemented.Thefirststep in thepreparationof thebindingsiteiscalculationofthesolventaccessiblesurfaceareaofthereceptor,without hydrogen atoms, using the probe radius of 1.4 Å. ThenegativeimageofthesurfaceisthencreatedbyoverlappingspheresusingSPHGENprogram.ThesphereswereconvertedtoPDBfilesformanualselection.Thefinalclusterusedfordockinghad91spheres.Subsequently, the gridswere calculated in a two stepprocedure.Firstly, the box around the binding site was constructed with theSHOWBOX program. Secondly, the grids were calculated with theaccessoryprogramcalledGRIDusing0.4gridspacing,9999Adistancecutoffanda4rdistancedependantdielectricconstant.Finally,virtualscreeningwascarriedbyloadingthenecessaryparameterintheinputfilewith“flexible_ligand”and“minimize_ligand”parametersturnedon.

PLANTS setupThedockingalgorithminPLANTSisbasedonaclassofstochasticoptimizationalgorithms called ant colony optimization. The docking in PLANTS wasconductedusingthestandardsettingsandPLANTSchemplpscoringfunction.ThebindingsitewascalculatedbyrunningthePLANTSinbindmode.Finally,allothernecessaryparametersweredefinedintheconfigurationfileandPLANTSwasruninscreenmode.

VINA setup ForVINAthereceptorfileandligandswereconvertedtoPDBQTformats using prepare_receptor4.py and prepare_ligand4.pyAutoDock scripts. The binding site coordinates were accepted

Figure 2 SchematicrepresentationofthestepsinvolvedinvirtualscreeningforidentifyingpotentialinhibitorsofB. anthracis thioredoxinreductase.

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ascalculatedbyPLANTSandtheconfigurationfilewaspreparedwithexhaustiveness8andcpuvalue4.

Results and discussionIdentification of proteins responsible for pathogenicity The BLASTP analysis identified 4459 orthologous proteinsfrom 8504, 6257 and 4937 unique protein sequences in B. cereus, B. anthracis and B. toyonensis, respectively (Figure 5). Additionally,inapairwisecomparison,itwasobservedthat5783protein sequenceswere common inB. anthracis andB. cereus (sequences present in pathogenic strains); 4636were commonin B. cereusandB. toyonensis while4497inB. anthracisandB. toyonensis. The subtraction of the total number of sequencespresentinthenon-pathogenicstrain,B. toyonensis(4937)fromthenumber of sequences present in pathogenic strains (5783)resultedin846proteinsequenceswhichmayplayanimportantroleinpathogenicity.Thenextstepinourapproachwastofilteroutsequencessimilartohumanproteins.Wefound43proteinsequencestobecloseenoughtohumanproteinsandhencewerediscardedresultingin803proteinsfrompathogenicstrainswithnosignificantsequencesimilaritytohumanproteins.

Figure 3 StructuralcomparisionofE. colithioredoxinreductase(EcTrxR)withB. anthracisthioredoxinreductase(BaTrxR).(AandB)

TheribbonandsurfacerepresentationoftheFR form of EcTrxR,respectively.(CandD)Theribbonandsurfacerepresentationof FO form of BaTrxR,respectively.IntheFRconformationtheTrxbindingregioniswideopenwhileintheFOconformationTrxbindingregionisonlypartiallyopen.

Identification of essential proteinsThe proteins involved in virulence and pathogenicity are veryimportant for the existence of pathogens. From 803 nonhomologous proteins we identified 10 essential proteins fromDEGdatabase (Table 1).This informationwasfurtherexploitedtopredictnoveldrugtargets.

Drug target identification and druggability assessmentDruggabilityisanimportantfeaturethatdescribesthepropertiesof proteins which enable interaction with high affinity andspecificitytosmalldrug-likemolecules.Alargenumberofproteinshave structural and physicochemical properties that hinderbinding to drugs thus rendering them “undruggable”. Currentapproachesforidentificationandevaluationofnoveldruggabletargetsaredescribedelsewhere[32].Oneoftheapproachesforevaluatingthedruggabilityofaproteinistoanalyzethesequencehomology to known drugs targets. Therefore, 10 essentialproteinsidentifiedabovewerealignedtothedrugtargetsintheDBdatabase.OnlyfourproteinswerefoundsimilartothebindingpartnersofFDAapproveddrugs,experimentalsmallmoleculesornutraceuticalcompoundspresentinDBdatabase.Thepercentage

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similarityofthefouressentialproteinswithDBtargets,organismname and availability of 3D structural information is shown inTable 2.However, it is important tonote that thisapproachoftargetidentificationdependsonlyontheproteinswithannotatedfunctions.Assuch,additionalfamiliesofdruggableproteinsmight

stillbeidentified.Amongthefouressentialproteinsthioredoxinreductase was shortlisted as the target of choice for dockinganalysisduetoseveralreasonsmentionedbelow:

The thioredoxin system including thioredoxin reductase,thioredoxin and NADPH are critical for DNA synthesis anddefenceagainstoxidative stress.Deoxyribonucleotides thatarebuildingblocksofDNAandaresynthesizedbyanenzymecalledribonucleotide reductase (RNR),which requires electrons fromeither thioredoxin or glutaredoxin (Grx) system [glutathionereductase (GR), glutathione (GSH), and Grx]. Interestingly, theGrx system ismissing inmany pathogenic bacteria such asM. tuberculosis, H. pylori, S. aureus, B. anthracis and B. cereus. ThismakesGrx-negativepathogens highly susceptible to drugstargetingbacterialTxrRsystem.Moreover,inotherbacteriasuchasM. tuberculosis [33],S. aureus [34]andN. gonorrhoeae [35],TrxRhasbeenshowntobeessentialforsurvival.Furthermore,incontrasttomammalianTrxR,thesubstrateTrxhastointeractdirectly with the dithiol-disulphide redox centre in the lowmolecular weight bacterial TrxR, which in inaccessible to thesubstrate in mammalian TrxR. Therefore, the protein-proteininteractionsiteinB. anthracisTrxRdiffersfromthemammaliancounterpartandcanbeconsideredforselectivetargeting.Finally,theavailabilityof theB. anthracisTrxRcrystal structure inPDBalsoimpactedourdecisiontoshortlistitforfurtheranalysis.

Docking of ligands with Thioredoxin reductaseWeusedthreedifferentopensourcedockingprogramstopredictcompoundswithhighbindingaffinitytoBaTrxR.Theimportanceof Ala 223 and Asp 137 in binding thioredoxin to thioredoxinreductasehasbeendiscussedearlier.Theformationofhydrogenbondswiththeatomsofthesetworesiduesbyanappropriatelyplaced ligand could lead to ligand occupying the binding cleft

Figure 4 BindingsiteinteractionofE. coliThioredoxin(EcTrx)withE. coliThioredoxinreductase(EcTrxR).Thebindingischaracterized

byhydrogenbondsbetweenArg73andIle75ofEcTrxwithAla237andAsp139ofEcTrxR.

Figure 5 Venndiagramto illustratetheintersectionamongtheproteomesofthepathogenicBacillus speciesunder study. Comparison of protein sequencesreveals that there are 4459 (33%) orthologousproteinsequencescommontoallthethreespecies. In a pairwise comparison B. anthracis and B. toyonenisisshare67%,B. cereusandB. toyonensis share64%whileB. anthracis andB. cereus share50%oftheproteins.

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UniProt ID Name of the query protein

Name of the DEG homolog Homologs in DEG e-value Score

I0D5Y6 DNAreplicationprotein DNAbiosynthesisprotein 6 5.1e-12 160

I0D343 Thioredoxinreductase Thioredoxinreductase 5 7.5e-14 177

I0DAS0 RibosomalproteinL31typeB RibosomalproteinL31 4 1.9e-33 199

I0D429 MembraneproteinProbableconservedintegralmembrane

protein3 8.2e-12 159

I0D0H6 Carbonstarvationprotein

Carbonstarvationprotein 2 2.9e-16 200

I0D2E2 5-dehydro-2-deoxygluconokinase

Ribokinase-likedomain-containing

protein2 2.2e-14 182

I0D5Y2 C-5cytosinespecificDNAmethylase

DNA-methyltransferase(cytosine-specific) 2 1.6e-14 182

I0CXM4 Beta-fructofuranosidase

Sucrose-6-phosphatehydrolase/Beta-fructofuranosidase

1 2.6e-73 692

I0D2C8 Aminoacyl-histidinedipeptidase

Aminoacyl-histidinedipeptidase 1 4.3e-84 785

I0D2B8 Transcriptionalregulator,MarRfamily

MarRfamilytranscriptional

regulator1 3.4e-20 228

Table 1: EssentialproteinsofB. anthraciswiththeircorrespondingDatabaseofEssentialGenes(DEG)homolog,e-valueandscore.TheproteinsarearrangedinthedecreasingorderofnumberofhomologsintheDEGdatabase.

S.no UniProt ID Protein name DB target DB organism % similarity to DB

target Related PDB id % similarity to PDB

1. I0CXM4 Beta-fructofuranosidase Beta-fructosidase Thermotoga maritima 35(2E-67) 1UYP 35(2E-65)

2. I0D5Y2C-5cytosine-specificDNAmethylasefamily

protein

ModificationmethylaseHhaI

Haemophilus parahaemolyticus 27(1E-16) 3G7U 31(3E-13)

3. I0D2E2 2-keto-3-deoxy-gluconatekinase

2-keto-3-deoxy-gluconatekinase

Thermus thermophilus 26(1E-29) 2QCV 60(6E-141)

4. I0D343 Thioredoxinreductase Thioredoxinreductase

Staphylococcus aureus 21(3E-11) 4FK1 100(0.0)

TherelatedPDBidentifiersandthepercentageidentitytotheshortlistedessentialproteinsofB. anthracis isalsoreported.BLASTPanalysiswascarriedouttoinferthecorrespondingDBtargetsofthequeryproteins.

Table 2:NonhomologousessentialproteinsofBacillus anthracissimilartobindingpartnersofFDAapproveddrugsorexperimentalcompounds.

thereby blocking the attachment of the native substrate (Trx)toenzymeBaTrxR.Therefore,thecompoundsthatdidnotformhydrogenbondswitheitherAla223orAsp137residues inthePPIsiteofthereceptorwerenotconsideredfurtherforanalysis.We determined the hydrogen bond interaction pattern of thedocked ligands from FindHbond tool in UCSF Chimera. Therewere 37 compounds in DOCK, 57 in VINA and 215 in PLANTSthatsatisfiedthehydrogenbondcriterion.Beforeanalyzingthecommon ligands in the threedatasets, firstwe set a thresholddocking score as a filter to remove compounds predicted tohavelowbindingaffinity.Forcalculatingthethresholdscore,10starting compoundswere docked toBaTrxR receptor in all thedocking software individually. The threshold score was thensetwithin 10% limit of the average dock score of the startingcompounds.Outofthe37compoundsonly20compoundswereabovethethresholdscoreof-36inDOCKdataset.IncaseofVINA

andPLANTSdatasetswefoundthat28and65compoundsabovethethresholdscoreof-5.1kcal/moland-63respectively.Finally,wefoundfourcompoundscommoninthethreedockingresults.Thestructureofallthefourcompoundsin2Dformatisdisplayedin (Figure 6) and their Chemical formula, IUPAC names andcorrespondingPubChem identifiersare inTable 3. Thedockingscores for each ligand from three different docking programsare shown in (Table 4)while the respective docking poses aredisplayedinFigure 7.Wenotethatallthefourcompoundssharea similar scaffold which show a hydrogen bond interaction toeitherAla223orAsp137andareareasonablefittothepocketinbetween.TheligandswerealsostabilizedinsidetheactivesitethroughfavourablevanderWaalsandhydrophobicinteractionswith amino acid residues. Further optimization and testing ofthescaffoldand itsderivativesmayprovideamorepotentandselectiveBaTrxRinhibitorswithbetterdruggability.

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Figure 6 Structuresofligandsthatsatisfiedthethresholdlimitsandwerecommoninallthreedockingresults.

S.No. ZINC ID PubChem CID Chemical Formula IUPAC Name

1 ZINC00155351 736051 C18H19ClN2O2-chloro-N-(4-piperidin-1-

ylphenyl)benzamide

2 ZINC01393213 1480285 C17H16F2N2O2,6-difluoro-N-(4-pyrrolidin-

1-ylphenyl)benzamide3 ZINC20271252 28809802 C16H25N3 2,5-di(piperidin-1-yl)aniline

4 ZINC00360711 834706 C17H19FN2ON-[(2-fluorophenyl)methyl]-4-morpholin-4-ylaniline

Table 3: PubChemIds,ChemicalformulasandIUPACnamesoftheselectedfourcompoundsfoundcommoninallthreedockingprograms.

S.No ZINC ID DOCK Scores PLANTS Scores VINA binding energies (kcal/mol)

1 ZINC00155351 -40.91 -63.67 -6.22 ZINC01393213 -39.04 -63.54 -6.03 ZINC20271252 -37.73 -65.14 -5.44 ZINC00360711 -41.12 -71.82 -5.9

Table 4: Dockingresultsofthecompoundssatisfyingthethresholdscoresinthethreedockingprograms.

to validate the results of computational analysis in vitro. Thisstudy may serve as a template for designing novel inhibitorsforpathogenswherethioredoxinreductaseplaysakeyinredoxhomeostasis.

AcknowledgmentsWe thank Professor Alok Dhawan, Director, Institute of LifeSciences, Ahmedabad University for carefully reading themanuscript.

Conflict of InterestTheauthorsdeclarethattheyhavenoconflictofinterest.

ConclusionsToconclude,inthepresentstudy,proteomesofvariouspathogenicandnonpathogenic strains ofB. anthracis andB. cereus were comparatively analysed which resulted in stepwise selectionof ten proteins that can be targeted for effective drug designand development. Thioredoxin reductase was finally selectedas the most promising target candidate due to essentiality,druggabilityandavailabilityofX-raycrystallographicstructureinPDB.ThemoleculardockingstudieswithZINCcompounds intothe protein-protein interaction site of thioredoxin reductaseleadto the identificationofnovelpotential inhibitorssharingasimilar scaffold. Futureworkwill focus on further optimizationof thescaffoldandsynthesisofderivatives.Wewouldalso like

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Figure 7 SelectedligandsdockedtotheNADPHdomainofthetarget(4FK1). ThedockedposesinbrowncolourarefromUCSF

DOCKwhileposesinpinkcolourarefromPLANTSwhereasposesinskybluecolourposesarefromVINA.

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