Using Chemoinformatics, Bioinformatics, and Bioassay to Predict and Explain the Antibacterial

17 Pages·2017·0.65 MB·English

ORIGINAL ARTICLES
Using Chemoinformatics, Bioinformatics,
and Bioassay to Predict and Explain
the Antibacterial Activity of Nonantibiotic
Food and Drug Administration Drugs
Nour Aldin Kahlous,1 Muhammad Al Mohdi Bawarish,1 Keywords: antibiotics, virtual screening, docking, antibac-
Muhammad Arabi Sarhan,1 Manfred Ku¨pper,1 terial activity
Ali Hasaba,2 and Mazen Rajab1
1ArabInternationalUniversityFacultyofPharmacy,Daraa,
INTRODUCTION
SyrianArabRepublic. I
n May 2016, a nightmare had come true, after China,
2DepartmentofMedicalLaboratoryScience,University
researchers in the United States reported first case of re-
ofMassachusetts—Dartmouth,Massachusetts.
sistantbacteriathatovercameoneofthestrongestantibi-
otics found in our arsenal,1 which poses a real threat for
ABSTRACT global health. Unfortunately, finding new and effective anti-
Discovering of new and effective antibiotics is a major issue bioticsisverychallenging.Mainly,becauseofthefactthatmost
facing scientists today. Luckily, the development of computer oftheantibioticsarenaturalorsemisyntheticcompoundsthat
scienceoffersnewmethodstoovercomethisissue.Inthisstudy, comprisecomplexstructuresanduniquechemicalproperties,2
asetofcomputersoftwarewasusedtopredicttheantibacterial which make the process of finding compounds with these
activity of nonantibiotic Food and Drug Administration (FDA)- features very hard.3 Recently, the development of chemoin-
approveddrugs,andtoexplaintheiractionbypossiblebindingto formaticsandbioinformaticsmadeabreakthroughinthedis-
well-known bacterial protein targets, along with testing their coveryprocess,pavingthewaytovirtualscreening(VS)andits
antibacterial activity against Gram-positive and Gram-negative methods.4Ingeneral,twomainapproachesexistforVS,thefirst
bacteria. A three-dimensional virtual screening method that oneistheligand-basedvirtualscreening(LBVS),whichrelies
relies on chemical and shape similarity was applied using rapid onmolecularsimilaritytofindnewdrugs,5takingbenefitfrom
overlay of chemical structures (ROCS) software to select candi- the general assumption that similar compounds may have
date compounds from the FDA-approved drugs database that similarbiologicalactivity.6Thesecondapproachisstructure-
share similarity with 17 known antibiotics. Then, to check their basedvirtualscreening(SBVS),whichdependsontheknowl-
antibacterial activity, disk diffusion test was applied on Sta- edgeoftarget’sstructure and characteristics.7 The mostcom-
phylococcus aureus and Escherichia coli. Finally, a protein monly used method in SBVS is molecular docking.8 This
docking method was applied using HYBRID software to predict methodworksbymodelingtheinteractionbetweenachemical
the binding of the active candidate to the target receptor of its compoundandaproteinattheatomicleveltopredictthebe-
similarantibiotic.Ofthe1,991drugsthatwerescreened,34had haviorofthecompoundinthebindingsiteoftargetproteins.9,10
been selected and among them 10 drugs showed antibacterial In this study, LBVS was applied using rapid overlay of che-
activity, whereby drotaverine and metoclopramide activities mical structures (ROCS) software (ROCS 3.2.0.4; OpenEye Sci-
were without precedent reports. Furthermore, the docking pro- entificSoftware,SantaFe,NM;www.eyesopen.com),11whichis
cess predicted that diclofenac, drotaverine, (S)-flurbiprofen, (S)- a three-dimensional (3D) shape and chemistry comparison
ibuprofen, and indomethacin could bind to the protein target of softwarethatoverlaysthe3Dstructureandthechemicaltypefor
their similar antibiotics. Nevertheless, their antibacterial activi- everyatomofagivenmoleculewithadatabaseofmoleculesto
ties are weak compared with those of their similar antibiotics, findsimilarmolecules.
which can be potentiated further by performing chemical mod- This research used the Food and Drug Administration
ifications on their structure. (FDA)-approveddrugsasdatabaseasaresponsetothecallby
DOI:10.1089/adt.2016.771 ªMARYANNLIEBERT,INC. (cid:2) VOL.15 NO.3 (cid:2) APRIL2017 ASSAYandDrugDevelopmentTechnologies 89
KAHLOUS ET AL.
NIHassociatedirectorforsciencepolicytofindnewusesfor generateconformers,themaximumnumberofconformersto
thecurrentapproveddrugs.12 begeneratedwassetat200fortheFDAcompoundsdatabase
MostoftheFDA-approveddrugshavesimplestructureand and 40 for the selected antibiotics. Also, as the compounds
low molecular weight. Hence, the structure simplicity of the database was in canonical SMILES format, OMEGA was set
antibiotic and its broad antibacterial activity spectrum were to use its own torsion and ring libraries to identify and
consideredtoselectonerepresentativeantibioticfromeachof enumerate rotatable bonds and flexible rings.
12 antibiotic chemical families; except for quinolones, one By default, OMEGA does not apply a number of rotatable
antibiotic from each generation was included (the fourth bonds cutoff but it limits the amount of time spent generat-
generationwasnotinvestigated).Also,forbeta-lactams,one ingconformersforeachmoleculeby120s.Also,conformers
memberfromeachsubfamilywasconsidered. with strain energies >10kcal/mol threshold are rejected, and
Furthermore,clavulanicacidthatisabroad-spectrumbeta- minimum root mean square distance (RMSD) between two
lactamase inhibitor was included because evidence suggests conformers below which they are considered as duplicatesis
thatithasminimalantibacterialactivityinvitro.13 setat0.5A˚.
ToverifyROCSresults,diskdiffusionsusceptibilitytestwas In addition, OMEGA was able to create an ensemble of
applied according to Clinical Laboratory Standards Institute stereoisomers for compounds that had unspecified stereo-
documentM02-A12.14Nevertheless,theantibacterialactivity centers (up to five centers for each molecule) before passing
tests such as disk diffusion, minimum inhibitory concentra- the molecules for conformer generation. The previous step
tion,orminimumbactericidalconcentrationdonotgiveany was unnecessary to the antibiotics because the isomeric
explanation about the mechanism of action of antibacterial SMILESswereavailable.
activity for the tested compound. However, regarding this Third,ROCSwasusedtoscreentheresultedconformersofthe
study, the similarity between the active candidates and the compounds database to find compound conformers that are
antibioticsgivesagoodprobabilitythatcandidatescouldbind structurally similar to the selected antibiotics. The Tanimoto-
to the same antibiotic targets. Hence to prove that, the can- Comboscore,calculatedbyROCS,wasusedtorankthecandi-
didates have to be tested on isolated targets or use more ad- dates. This score is a combination of molecular volume
vancedmethods,whichwouldrequirespecialinstrumentsand similarity(shape)andchemicaltypesofatomssimilarity(color)
highskillsinadditiontohighcosts.15,16Luckily,thedevelop- betweentwocompounds,withafullmarkof2(1forshapeand1
mentofmoleculardockinghaspresentedafast,cheap,andeasy forcolor).Finally,thecandidatedrugswerechosenaccordingto
waytopredictthebindingofacompoundtoatargetreceptor.17 TanimotoComboscoreandtheiravailabilityinthelocalmarket.
Inthisstudy,OpenEyeHYBRID,18whichisaSBVSsoftware,
wasused(OEDOCKING3.2.0.2;OpenEyeScientificSoftware; List of Selected Antibiotics
www.eyesopen.com). HYBRID docks multiconformer mole- Ampicillin(CID:6249),azithromycin(CID:447043),cephradine
cules into a single or multiple protein receptors, relying on (CID38103),chloramphenicol(CID:5959),ciprofloxacin(CID:
systematic nonstochastic algorithm to predict possible pro- 2764), clavulanic acid (CID: 5280980), D-cycloserine (CID
tein–ligand poses within an active site. In addition, when a 6234), fusidic acid (CID: 3000226), gentamicin (CID: 3467),
protein–ligand complex crystal is available, HYBRID uses triclosan(CID:5564),levofloxacin(CID:149096),meropenem
boundligandinformationtoimprovedockingprocedure. (CID 441130), nalidixic acid (CID: 4421), sulfadiazine (CID:
5215), tetracycline (CID: 54675776), trimethoprim (CID:
MATERIALS AND METHODS
5578),vancomycin(CID:14969).
Chemoinformatics
First, the simplified molecular-input line-entry system Antibacterial Activity Test
(SMILES)forthe1,991FDA-approveddrug(asthecompounds To determine the antibacterial activity of the candi-
database) and 17 selected antibiotics (see List of Selected date drugs (List of Candidate Drugs section), the Kirby–
Antibiotics section) were extracted from DrugBank v4.5.0 Bauerdiskdiffusionsusceptibilitymethodwasused,which
approved drugs Structure Data Format file19 for the former is a simple and straight forward method to measure the
anddownloadedfromPubChem20forthelatter. activityofchemicalsbasedontheradiusofinhibitionzone
Second, the SMILESs of compounds were processed by surrounding the chemical loaded disk on the Mueller–
OpenEye OMEGA (OMEGA 2.5.1.4; OpenEye Scientific Soft- Hinton agarplate.21,22
ware, Santa Fe, NM. www.eyesopen.com. Hawkins, P.C.D.; The Kirby–Bauer disk diffusion susceptibility method pro-
Skillman, A.G.; Warren, G.L.; Ellingson, B.A.; Stahl, M.T.) to tocolwasappliedaccordingtoClinicalLaboratoryStandards
90 ASSAYandDrugDevelopmentTechnologies APRIL2017 ªMARYANNLIEBERT,INC.
ANTIBACTERIAL ACTIVITY OF NONANTIBIOTIC FDA DRUGS
InstitutedocumentM02-A12,14,20barringtheexceptionofthe NaOH 5%:50mg allopurinol (Zhejiang Ruiqing Pharma-
nonantibioticdiffusiondiskpreparationstep. ceutical,India),50mgfurosemide(AMRI,India).
First, the 4mm thick Mueller–Hinton agar (HiMedia Lab- Distilled water:25mg betahistine (Kinfonpharma, China),
oratories,India)waspreparedin90·15-mmplates(Citotest, 50mganhydrouscaffeine(BASF,Germany),50mgcaptopril
China) according to the manufacture recipe. Second, topre- (Shandong Weifang Pharmaceutical, China), 50mg guaife-
pare disks containing 500 and 1,000mg from each drug, a nesin(GranulesIndiaLimited,India),50mglidocaine(Neha
specific amount of each drug was dissolved in 1mL of suit- PharmaPvt.Ltd.,India),50mgmetformin(AartiDrugsLtd.),
able solvent (acetone, methanol, ethanol from ChamLab, 50mg metoclopramide (SL Drugs and Pharmaceuticals, In-
Syria;ordistilledwater)(seetheListofCandidateDrugswith dia), 50mg niacin (Aarti Drugs Ltd.), 50mg ondansetron
Their Solubility section). Third, specific volumes from each (Shodana,India),50mgphenylephrine(TajPharmaceuticals
drug solution were loaded on 6mm blank diffusion disks Limited),50mgpiracetam(JiangxiYuehuaPharmaceutical,
(Whatman, United Kingdom) using micropipettes (Dragon China), 25mg pregabalin (Changzhou Pharmaceutical Fac-
LaboratoryInstruments,China)andweredriedat37(cid:2)C.Also, tory), 50mg pyridoxine hydrochloride (Excellent Health
to rule out any antibacterial effect that results from the sol- Products Co., Ltd., China), 50mg thiamine hydrochloride
vents,controldisksfromeachsolventwereprepared.Fourth, (ExcellentHealthProductsCo.,Ltd.).
theagarplateswereinoculatedseparatelywith0.5McFarland
suspension of Escherichia coli (identified microscopically
List of Reference Antibiotic Disks
byGramstain,eosinmethyleneblueagarfromHiMediaLa-
and Their Load of Antibiotic
boratories23), and Staphylococcus aureus (identified micro-
Azithromycin 15mg, meropenem 10mg, nalidixic acid 30mg,
scopicallybyGramstain,catalase,andcoagulasetests23)was
and tetracycline 30mg from Bioanalyse (Turkey). Chloramph-
takenfromthemicrobiologylaboratoryinArabInternational
enicol 30mg, ciprofloxacin 5mg, gentamicin 10mg, and levo-
University, and the prepared drug disks plus the reference
floxacin5mgfromAbtek(UnitedKingdom).
antibiotic disks (see List of Reference Antibiotic Disks and
Their Load of Antibiotic section) were placed on the inocu-
lated plates. Finally, the plates were incubated for 24h at Molecular Docking Protocol
37(cid:2)C and the zones of inhibition were measured for each First, the complex of the antibiotic or its relative (same
candidatedrug(Table1). chemicalfamily)anditstargetreceptorwasdownloadedfrom
ProteinDataBank(PDB)www.rcsb.org.24Then,thereceptors
List of Candidate Drugs with Their Solubility (List of Crystal Structures of Protein–Ligand Complexes sec-
in 1mL of Solvent tion) were prepared for docking by OEDOCKING Make Re-
Acetone 98%:25mg Hydrochlorothiazide (Changzhou ceptor (version 3.2.0.2) tool using the default parameters
PharmaceuticalFactory,China). except forsetting SER1048B as donor bindingconstraintfor
Ethanol 98%:50mg aspirin (Novacyl, France), 50mg di- 2XCTreceptor.Afterthat,theconformersforbothantibiotics
clofenac sodium (Aarti Drugs Ltd., India), 50mg drotaverine and active candidates were generated from their isomeric
hydrochloride(RaChemPharmaLtd.,India),50mgephedrine SMILES by OMEGA by setting the number of conformers at
(TajPharmaceuticalsLimited,India),50mgflurbiprofen(FDC 200, taking into consideration the protonation and the ste-
Limited, India), 50mg ibuprofen (Shasun, India), 50mg in- reoisomerismstatesforthecompoundsiftheyexist.Next,the
dapamide (Ami Life Sciences Pvt. Ltd., India), 25mg indo- docking process was performed by OpenEye HYBRID using
methacin (AS Joshi & Company, India), 50mg ketoprofen the default parameters except for the docking resolution
(YibinHopePharmaceutical,China),12mglamotrigine(Divi’s parameter it was set at high mode. HYBRID performs ex-
Laboratories Limited, India), 50mg pseudoephedrine (Taj haustive search by rotating and translating the rigid con-
Pharmaceuticals Limited), 50mg salbutamol sulfate (FDC former within the active site with a Chemgauss4 scoring
Limited), 50mg salicylic acid (Merck KGaA, Germany), and function. The Chemgauss4 scoring function uses smooth
50mgwarfarinsodium(HubeiOceanBiotechCo.,Ltd.,China). Gaussian functions to represent the shape and chemistry of
Ammonia 25%:50mg theophylline (Taj Pharmaceuticals moleculesandisawareofinteractionssuchasshape(contact),
Limited). hydrogen bonds (acceptor anddonor),chelating (metal),and
Methanol 98%:25mg clopidogrel bisulphate (IOL Chemi- desolvation.
cals and Pharmaceuticals Limited, India), 50mg gliclazide ThedockingtypeperformedbyHYBRIDisrigidandthereby
(ZhejiangJiuzhouPharmaceuticalCo.,Ltd.,China). cannotassignflexiblechainsinreceptors.
ªMARYANNLIEBERT,INC. (cid:2) VOL.15 NO.3 (cid:2) APRIL2017 ASSAYandDrugDevelopmentTechnologies 91
KAHLOUS ET AL.
Table 1. Compound Similarities and Antibacterial Activities Results
Compounds Similarities (Tanimoto) Antibacterial Activitiesa (mm)
Reference Candidate Combo(out Shape (out Color(out Escherichia Staphylococcus
Antibiotic Drug oftwo) ofone) ofone) coli aureus
Amphenicols
Chloramphenicol 12b,c 24c,d
Salbutamol 1.104 0.749 0.355 - -
Aminoglycosides
Gentamicin - - - - 17c,e 19b,c
Beta-lactamderivatives
Ampicillin f f
Ketoprofen 1.183 0.734 0.449 - -
Indapamide 1.076 0.762 0.314 - -
Gliclazide 1.008 0.712 0.296 - -
Cephradine f f
Ketoprofen 1.038 0.712 0.326 - -
Indapamide 1.07 0.79 0.28 - -
Gliclazide 0.975 0.636 0.338 - -
Clavulanicacid f f
Aspirin 1.491 0.851 0.64 - -
Captopril 1.315 0.762 0.553 - 7/8
Niacin 1.277 0.675 0.602 - 7/8
Pyridoxine 1.205 0.781 0.424 - -
Betahistine 1.106 0.693 0.413 - -
Pregabalin 1.064 0.802 0.262 - -
Meropenem 32c,d 32c,d
Furosemide 1.119 0.752 0.367 - -
D-Alanineanalogue
D-Cycloserine f f
Allopurinol 1.128 0.821 0.307 - 7/8
Piracetam 1.101 0.711 0.39 - -
Metformin 1.001 0.743 0.258 - -
Diphenyletherderivative
Triclosan f f
Guaifenesin 1.321 0.832 0.489 - -
(continued)
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Table 1. (Continued)
Compounds Similarities (Tanimoto) Antibacterial Activitiesa(mm)
Reference Candidate Combo(out Shape (out Color(out Escherichia Staphylococcus
Antibiotic Drug oftwo) ofone) ofone) coli aureus
Warfarin 1.185 0.697 0.488 - -
Pseudoephedrine 1.173 0.745 0.428 - -
Lamotrigine 1.159 0.787 0.372 7/8 -
Ephedrine 1.139 0.765 0.374 - -
Diclofenac 1.067 0.855 0.212 - 14/18
Phenylephrine 1.029 0.74 0.289 - -
Lidocaine 1.024 0.755 0.269 - -
Fusidane
Fusidicacid - - - - f f
Glycopeptides
Vancomycin - - - - f f
Macrolides
Azithromycin - - - 18c,h 30c,d
Pyrimidinederivative
Trimethoprim f f
Thiamine 1.329 0.804 0.525 - -
Quinolonesandfluoroquinolones
Ciprofloxacin 12b,c 25c,d
Indomethacin 1.082 0.744 0.338 - 9/10
Drotaverineg 0.91 0.68 0.23 - 13/15
Levofloxacin 9b,c 28c,d
Ondansetron 1.081 0.775 0.306 - -
Drotaverine 1.04 0.747 0.293 - 13/15
Nalidixicacid 13b,c 13c,h
Caffeine 1.25 0.826 0.424 - -
Salicylicacid 1.281 0.662 0.619 - -
Theophylline 1.207 0.825 0.382 - -
Ibuprofen 1.083 0.696 0.387 - 9/10
Hydrochloro-thiazide 1.038 0.727 0.311 - -
Flurbiprofen 1.019 0.649 0.37 - 11/12
Diclofenacg 1.003 0.592 0.411 - 14/18
(continued)
ªMARYANNLIEBERT,INC. (cid:2) VOL.15 NO.3 (cid:2) APRIL2017 ASSAYandDrugDevelopmentTechnologies 93
KAHLOUS ET AL.
Table 1. (Continued)
Compounds Similarities (Tanimoto) Antibacterial Activitiesa (mm)
Reference Candidate Combo(out Shape (out Color(out Escherichia Staphylococcus
Antibiotic Drug oftwo) ofone) ofone) coli aureus
Sulfonamides
Sulfadiazine f f
Metoclopramide 1.119 0.795 0.324 - 8/9
Clopidogrel 1.008 0.784 0.224 - -
Tetracyclines
Tetracycline - - - - 11c,h 27c,d
Thebacterialsusceptibilityprofile,againstantibiotics,wasretrievedfrommanufacturingcompanies.
aCandidate’saverageinhibitionzoneinmillimeter(mm)at500/1000mg.Thestandarddeviationsofallmeasurementswerebetween1.41and0.70mm.
bResistant.
cUsedinstandardizeddiskform(seeListofReferenceAntibioticDisksandTheirLoadofAntibioticsectionforquantities).
dSusceptible.
eIntermediate.
fThecompoundisnotavailablefortest.
gSetfordockingpurpose.
hUnknownbecausethebacteriasusceptibilityagainsttheseantibioticshasnotbeensetbythemanufacturingcompany.
-,negative.
Finally, OpenEye VIDA 4.3.0.4 was used to show the pre- V, and W, each one has a length of eight residues. Four
dictedbindingposeinthereceptor-bindingpocketcompared polydeoxyribonucleotides: G, H, X, and Y, each one has a
withthebindingposeofthecrystalligand. length of 12 residues and in complex with ciprofloxacin.
EachofthechainsB,D,F,G,S,U,andWbindsmanganese
List of Crystal Structures of Protein–Ligand Complexes ion (Mn2+).
2XCS: ‘‘The 2.1 A˚ crystal structure of S. aureus gyrase 4ALI:‘‘CrystalstructureofS.aureusFabIincomplexwith
complex with GSK299423 and DNA.’’25 Resolution: 3.35 A˚, NADPandtriclosan(P1).’’26Resolution:2.1A˚.Proteinname:
protein name: DNA gyrase, type II topoisomerase (catalytic NAD(P)H-dependenttrans-2-enoylacylcarrierprotein(ACP)
activity: ATP-dependent breakage, passage, and rejoining of reductase.Oxidoreductase.(Catalyticactivity:anacyl-[ACP]+
double-stranded DNA). Gene names: gyrB (Ordered Locus NADP+=a trans-2,3-dehydroacyl-[ACP]+NADPH). Gene
Name:SA0005)andgyrA(OrderedLocusName:SA0006). name: fabI (Ordered Locus Names: b1288, JW1281).
Description: The structure 2XCS has in total four chains. Description:Thestructure4ALIhasintotaleightidentical
Twoidenticalpolypeptide(L)s:B,Deachonehaslengthof692 polypeptide(L)chains:A,B,C,D,E,F,G,andH,eachchainhas
residuesthatformtwosubunitsAandBofDNAgyrase.Two alengthof282residuesandisincomplexwithtriclosanand
polydeoxyribonucleotidesEandF,eachonehaslengthof20 NADP+.EachofthechainsA,B,C,E,F,andGisincomplex
residues.ChainsF,E,B,andDincomplexwithGSK299423. withglutamicacid.
2XCT: ‘‘The twinned 3.35 A˚ structure of S. aureus gyrase
complexwithciprofloxacinandDNA.’’25Resolution:3.35A˚. RESULTS
Protein name: DNA gyrase, type II topoisomerase (catalytic To find drugs similar to the selected 17 antibiotics, the
activity: ATP-dependent breakage, passage, and rejoining compounds of FDA-approved drug database that have Tani-
ofdouble-strandedDNA).Genenames:gyrB(OrderedLocus motoCombo score equal to or >1 and available from the
Name:SA0005)gyrA(OrderedLocusName:SA0006). commercial vendors were considered as candidates in our
Description: The structure 2XCT has in total 12 chains. investigation. As shown in Table 1 (or Supplementary
Fouridenticalpolypeptides(L):B,D,S,andU,eachonehasa Table S1; Supplementary Data are available online at www
length of 692 residues that form four subunits: two A and .liebertpub.com/adt), 34 candidates similar to 12 antibiotics
two B of DNA gyrase. Four polydeoxyribonucleotides: E, F, wentthroughtheantibacterialtest.
94 ASSAYandDrugDevelopmentTechnologies APRIL2017 ªMARYANNLIEBERT,INC.
ANTIBACTERIAL ACTIVITY OF NONANTIBIOTIC FDA DRUGS
E. coli has resisted four reference antibiotics (chloram- active candidates were docked on this new site. Moreover,
phenicol,ciprofloxacin,levofloxacin,and,nalidixicacid)and diclofenac showed the best antibacterial action between
S. aureus is susceptible to all tested reference antibiotics the candidates, which is worth to be investigated more. As
exceptforgentamicin. showninTable1,diclofenacsharessimilaritywithtriclosan.
Furthermore,E. coli alsoresisted all thecandidates except Therefore, docking it to triclosan receptor may give a dif-
for mild effect showed by lamotrigine (anticonvulsant). In ferentexplanationtoitsactivity.
contrast, nine compounds showed activity toward S. aureus. Next, to elaborate the understanding of activity observed,
Theactivecandidatesrankedbytheirinhibitionzonesizeare the docking of most potent antibacterial activity candi-
diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), dates, drotaverine and diclofenac, was examined using
drotaverine(antispasmodic),flurbiprofen(NSAID),ibuprofen OpenEyeVIDA.
(NSAID), indomethacin (NSAID), metoclopramide (antiemet- The three quinolone antibiotics and the active candidates
ic),niacin(vitaminB ),allopurinol(uricacidloweringagent), were docked to all sites in 2XCS (one site) and 2XCT (four
3
andcaptopril(antihypertensive). sites).Onlytriclosananddiclofenacweredockedtoallsitesin
AsshowninTable 1,ROCSdidnotfindanynonantibiotic 4ALI(eightsites).Foreachcrystal,theresultsofsitewithbest
FDA-approveddrugsimilartoazithromycin,fusidicacid,gen- HYBRIDscoreswereretained.
tamicin,tetracycline,andvancomycin.Mainly,becauseallthe Minimum RMSDs between the ligand in crystal and the
TanimotoCombo scores calculated between those antibiotics HYBRID’sdockedligand(retrievedfromthecrystal)were0.31,
and the nonantibiotic FDA-approved drugs were <1. There- 0.75,and0.6A˚ forGSK299423,ciprofloxacin,andtriclosan,
fore,thisconfirmsthatthoseantibioticshaveuniquechemical respectively (Figs. 1 and 2). Hence, the rigid docking using
structures(SupplementaryTableS1)asmentionedpreviously HYBRIDcouldreflectpossiblemolecularligandpositioninto
intheintroduction. theactivesite.28
Ingeneral,rankingofcompoundsbyROCSTanimotoCombo In general, when the active candidates were docked to
scorewasdifferentthantheirinhibitionzone’sranking.Hence, 2XCT,theirHYBRIDscoreswerebetterthantheirequivalents
anegativeassociationbetweenscoreandantibacterialactivity with2XCS.Hence,onlydockingof2XCTresultswasretained.
couldbenoted,suchasthiaminehavinggoodscorebutfailedin Table2representsthedockingresultsofthethreequinolone
antibacterial test or when indomethacin, drotaverine, flurbi- antibiotics and their similar active candidates to 2XCT crys-
profen,ibuprofen,anddiclofenachavelowerTanimotoCombo tal complex25 S. aureus gyrase with ciprofloxacin and DNA
scoresthantheothercandidatesbutcameupwithgoodresults complex.ItisworthmentioningthattheminimumRMSDbe-
inantibacterialtest.Nevertheless,somecompoundswithrela- tweenciprofloxacinincrystalandtheconformergeneratedfor
tivelyhighscoresuchascaptoprilandniacinhaveadetectable protonatedciprofloxacinusingthedefaultconformergenerator
antibacterial effect and others laid on the score edge of the libraryofOMEGAwas1.52A˚,withdifferenceintheorientation
cutoff such as clopidogrel, gliclazide, and lidocaine had no ofthe–COO-groupthatpreventedthegeneratedconformerof
antibacterialactivityatall. protonated ciprofloxacin from forming a chelator bond with
To give a possible explanation for the antibacterial ac- Mn2+inthe2XCTcrystal.Therefore,protonatedciprofloxacin
tivity showed by the active candidates, OpenEye HYBRID inthe2XCTcrystalwasusedasabaseofbondcomparison.
was used to dock the candidates that achieved an inhibi- Comparing with the protonated ciprofloxacin in the 2XCT
tion zone ‡10mm, and they were indomethacin, drotaver- crystal, all compounds have made a hydrogen bond with
ine, ibuprofen, flurbiprofen, and diclofenac. In fact, those SER1084Bexceptforprotonateddrotaverine.Likewise,allthe
antibacterial active candidates were found to be similar to compoundsformachelatingbondwithMN2000Gexceptfor
ciprofloxacin, levofloxacin, and nalidixic acid, which all drotaverine(neutralorprotonated),protonatedindomethacin,
belong to quinolones’s chemical family that targets DNA (S)-flurbiprofen, and (S)-ibuprofen. Moreover, among all the
gyrase.27Thus,thedockingcanbedoneononereceptorsite candidates,onlydrotaverine(neutralorprotonated)hasmade
forall.However,Baxetal.discoveredthatDNAgyrasecould a hydrogen bond with ARG458B. Unlike ciprofloxacin in
be inhibited by interacting with another site other than the thecrystal,(S)-flurbiprofen,(S)-ibuprofen,andindomethacin
classical site of quinolones,25 where authors demonstrated formed hydrogen bond with ARG1122D. Finally, regarding
that the inhibitor (GSK299423) was located between the the interactions with nucleotides, neutral drotaverine and
DNAandatransientnoncatalyticcavityonthetwofoldaxis (S)-flurbiprofen made a hydrogen bond with DG9G, levo-
at the DNA gyrase Adimerinterface, andwas closedtothe floxacin, ciprofloxacin, and nalidixic acid form a hydrogen
active sites and fluoroquinolone binding sites.25 Hence, bondwithDT8E.
ªMARYANNLIEBERT,INC. (cid:2) VOL.15 NO.3 (cid:2) APRIL2017 ASSAYandDrugDevelopmentTechnologies 95
Fig.1. Clusterof10bestdockingposes(transparentred)ofciprofloxacinin2XCTcrystal.(A)Dockedciprofloxacinwasretrievedfromcrystal.
(B)DockedciprofloxacinwasgeneratedbythedefaultconformergeneratorlibraryofOMEGA.Theciprofloxacin(black)ispartofthe2XCT
crystal.Forclaritypurpose,ineachpanel,oneaminoacidandonenucleotidearedisplayedinboxeswithouttheirchemicalstructures.
96 ASSAYandDrugDevelopmentTechnologies APRIL2017 ªMARYANNLIEBERT,INC.
Fig.2. Clusterof10bestdockingposes(transparentred)oftriclosanin4ALIcrystal.(A)Dockedtriclosanwasretrievedfromcrystal.(B)
DockedtriclosangeneratedbythedefaultconformergeneratorlibraryofOMEGA.Thetriclosan(black)ispartofthe4ALIcrystal.Forclarity
purpose, some ofamino acidsare displayedin boxes without their chemicalstructures.
ªMARYANNLIEBERT,INC. (cid:2) VOL.15 NO.3 (cid:2) APRIL2017 ASSAYandDrugDevelopmentTechnologies 97
KAHLOUS ET AL.
Table 2. Candidates Binding Mode Predicted by Hybrid in Comparison with Protonated Ciprofloxacin in 2XCT Crystal
Compound Acceptor Donor Metala Contactb
Levofloxacin DT8E SER1084B,ARG458B MN2000G ASP437B,DA13H,DC12H,DG9G,GLY459B
Levofloxacinc SER1084B,ARG458B MN2000G DA13H,DC12H,DG9G,DT8E,GLY459B
Ciprofloxacinc(crystal) SER1084B,ARG458B MN2000G DA13H,DC12H,DG9G,DT8E,GLY459B
Ciprofloxacin DT8E SER1084B,ARG458B MN2000G DA13H,DC12H,DG9G,GLY459B
Ciprofloxacinc,d SER1084B ARG458B,DA13H,DC12H,DG9G,DT8E,GLY459B,MN2000G
Nalidixicacidc SER1084B MN2000G ARG437B,ARG458B,DA13H,DG9G,DT8E,GLY459B
Indomethacinc SER1084B ARG437B,ARG458B,DA13H,DG9G,DT8E
Flurbiprofene SER1084B,ARG1122D,DG9G ARG458B,DA13H,DT8E,GLY459B,MN2000G
Indomethacin SER1084B,ARG1122D MN2000G ARG458B,DA13H,DT8E,GLY1082B,GLY459B,MN2000G
Flurbiprofenc,e SER1084B MN2000G ARG458B,DA13H,DC12H,DG9G,DT8E,GLY459B
Nalidixicacid DT8E SER1084B MN2000G ARG458B,DA13H,DG9G,GLY459B
Ibuprofene SER1084B,ARG1122D ARG458B,DA13H,DG9G,DT8E,GLY459B
Diclofenac SER1084B MN2000G ARG437B,ARG458B,DA13H,DG9G,DT10G,DT8E,GLY459B
Drotaverinec ARG458B ASN476B,DA13H,DC12H,DG9G,DT8E,GLU477B,GLY1082B,
GLY459B,MN2000G,SER1084B,SER1085B
Drotaverine SER1084B,ARG458B,DG9G ASN476B,DA13H,DC12H,DT10G,DT8E,GLU477B,
GLY1082B,MN2000G,SER1085B
Ibuprofenc,e SER1084B MN2000G ARG458B,DA13H,DC12H,DG9G,DT8E
Diclofenacc SER1084B MN2000G ARG458B,ARG473B,DA13H,DG9G,DT8E,GLY459B
Candidatesarearrangedbydescendingorderofhybridscore.
aMetal–chelatorinteraction.
bLigandheavyatomiswithin4A˚ oftheproteinheavyatom.
cProtonated.
dBecausethedefaultconformergeneratorlibraryofOMEGAwasused,theconformergeneratedforprotonatedciprofloxacinwasdifferentfromtheprotonated
ciprofloxacininthe2XCTcrystal.
eOnlytheresultsoftheSisomersareshownbecausetheresultsofRisomersareweakandinsignificanttoshow.
The binding model of drotaverine, ciprofloxacin, and Table 3 represents the docking of triclosan and diclofenac
gyrase-DNA is depicted in Figure 3, which revealed that (neutralorprotonated)tothesiteofenoyl-ACPreductasefrom
drotaverinefitswellintheciprofloxacinbindingpocketandis S. aureus based on enoyl-ACP reductase–NADP+-glutamate-
evenmoreclosetonucleotideDT10Gandaminoacidssuchas triclosancomplexstructure;4ALIcrystal.26Itisworthtonote
ASN476B, GLY1082B, GLU477B, and SER1085B. Similar to that the RMSD of the triclosan conformer generated by the
ciprofloxacin, drotaverine forms two hydrogen bonds with default conformer generator library of OMEGA was 0.39 A˚,
SER1084B (2.94 A˚) and ARG458B (2.3 A˚), and two p–p in- which is near the RMSD of the docked triclosan (retrieved
teractions between aromatic ring in ligand and pyrimidine fromcrystal).
ring in DG9G (3.67 A˚), and DA8E (3.55 A˚). Contrary to ci- Obviously,triclosananddiclofenac(neutralorprotonated)
profloxacin, drotaverine has one different hydrogen bond bindtothereceptorbutwithsomedifferencesininteractions.
withDG9G(3.10A˚).Also,4-carbonyland3-carboxylgroups First, diclofenac (neutral or protonated) binds to SER197H
inciprofloxacinanchorMn2+,but,eventhatMn2+iscloseto whereastriclosanbindstoTYR157H.Second,thethreecom-
drotaverine(3.55A˚),thesebondsareabsent. poundsbindtoNAP1257Hbutwithdifferentbindingmodes.
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