Discovery of an Inhibitor of Z-Alpha1 Antitrypsin Polymerization

18 Pages·2015·2.63 MB·English

RESEARCHARTICLE
Discovery of an Inhibitor of Z-Alpha1
Antitrypsin Polymerization
ValerieBerthelier1,2☯*,JasonBrettHarris2,4☯¤,KaseyNoelEstenson1,2,
JeromeBaudry2,3,4*
1 DepartmentofMedicine,UniversityofTennesseeHealthScienceCenter—GraduateSchoolofMedicine,
Knoxville,Tennessee,UnitedStatesofAmerica,2 UT-ORNLGraduateSchoolofGenomeScienceand
Technology,UniversityofTennessee,Knoxville,Tennessee,UnitedStatesofAmerica,3 Departmentof
BiochemistryandCellularandMolecularBiology,UniversityofTennessee,Knoxville,Tennessee,United
StatesofAmerica,4 UT-ORNLCenterforMolecularBiophysics,OakRidgeNationalLaboratory,OakRidge,
Tennessee,UnitedStatesofAmerica
a11111
☯Theseauthorscontributedequallytothiswork.
¤ Currentaddress:USEnvironmentalProtectionAgency,NationalCenterforComputationalToxicology,
ResearchTrianglePark,NorthCarolina,UnitedStatesofAmerica
* [email protected](VB); [email protected](JB)
Abstract
OPENACCESS
Citation:BerthelierV,HarrisJB,EstensonKN, PolymerizationoftheZvariantalpha-1-antitrypsin(Z-α1AT)resultsinthemostcommon
BaudryJ(2015)DiscoveryofanInhibitorofZ-Alpha1
andsevereformofα1ATdeficiency(α1ATD),adebilitatinggeneticdisorderwhoseclinical
AntitrypsinPolymerization.PLoSONE10(5):
e0126256.doi:10.1371/journal.pone.0126256 manifestationsrangefromasymptomatictofatalliverand/orlungdisease.Asthealtered
conformationofZ-α1ATanditsattendantaggregationareresponsibleforpathogenesis,the
AcademicEditor:PaoloCarloni,Computational
Biophysics,GERMANY polymerizationprocesspersehasbecomeamajortargetforthedevelopmentoftherapeu-
tics.BasedontheabilityofZ-α1ATtoaggregatebyrecruitingthereactivecenterloop
Received:December16,2014
(RCL)ofanotherZ-α1ATintoitss4Acavity,wedevelopedahigh-throughputscreening
Accepted:March31,2015
assaythatusesamodified6-merpeptidemimickingtheRCLtoscreenforinhibitorsofZ-
Published:May11,2015 α1ATpolymergrowth.AsubsetofcompoundsfromtheLibraryofPharmacologicallyActive
Copyright:©2015Berthelieretal.Thisisanopen Compounds(LOPAC)withmolecularweightsrangingfrom300to700Da,wasusedto
accessarticledistributedunderthetermsofthe evaluatetheassay’scapabilities.TheinhibitorS-(4-nitrobenzyl)-6-thioguanosinewasiden-
CreativeCommonsAttributionLicense,whichpermits
tifiedasaleadcompoundanditsabilitytopreventZ-α1ATpolymerizationconfirmedbysec-
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare ondaryassays.TofurtherinvestigatethebindinglocationofS-(4-nitrobenzyl)-6-
credited. thioguanosine,aninsilicostrategywaspursuedandtheintermediateα1ATM*statemod-
DataAvailabilityStatement:Allrelevantdataare eledtoallowmoleculardockingsimulationsandexplorevariouspotentialbindingsites.
withinthepaperanditsSupportingInformationfiles. DockingresultspredictthatS-(4-nitrobenzyl)-6-thioguanosinecanbindatthes4Acavity
Funding:ThisworkwassupportedbyanAlpha-1 andattheedgeofβ-sheetA.TheformerbindingsitewoulddirectlyblockRCLinsertion
Foundation'sgrant[http://www.alpha1portal.org]to whereasthelattersitewouldpreventβ-sheetAfromexpandingbetweens3A/s5A,andthus
VB,andagrantfromThePhysicians’Medical
indirectlyimpedeRCLinsertion.Altogether,ourinvestigationshaverevealedanovelcom-
EducationandResearchFoundation[http://www.
utmedicalcenter.org/for-health-professionals/ poundthatinhibitstheformationofZ-α1ATpolymers,aswellasinvitroandinsilicostrate-
resources/physicians-medical-education-and- giesforidentifyingandcharacterizingadditionalblockingmoleculesofZ-
research-foundation/]toVB.Thefundershadnorole α1ATpolymerization.
instudydesign,datacollectionandanalysis,decision
topublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSONE|DOI:10.1371/journal.pone.0126256 May11,2015 1/18
Z-α1ATPolymerizationInhibitor
Introduction
Humanα1-antitrypsin(α1AT)isthemostabundantmemberoftheserineproteaseinhibitor
(SERPIN)family.Itisasoluble52-kDaglycoproteinsynthesizedprimarilybyhepatocytesand
deliveredtothelungstoaccomplishitscriticalfunction:inactivationoftheproteinaseneutro-
philelastase(NE),amediatorofalveolardestruction[1].Defectivefolding,traffickingandse-
cretionintotheplasmaofα1ATareresponsibleforα1ATdeficiency(α1ATD)[2,3].
Thestructuralflexibilityofα1ATisimportantforittoperformitsanti-proteasefunction
andensurelungintegrity.Withacoredomaincomposedof3β-sheetsA,BandC,and9α-he-
lices,α1ATfeaturesanexposedandflexiblereactivecenterloop(RCL)thatservesasbaitfor
NE.Uponbindingtotheproteinase,adramaticconformationalchangeoccursasRCLis
cleavedandtranslocatesintoβ-sheetAtoformthenewcentralandfourthstrand,s4A.The
translocationeventcarriesalongNEfromonesidetotheotherofα1AT,causingitsinactiva-
tionbyforminganirreversible,highermolecularweightsuicidecomplex[4,5].Areductionor
lackofthisinhibitionthroughloop-sheetinsertionandproteolyticcleavageisthoughttobe
theunderlyingmechanismresponsibleforα1ATD[6,7].
Over100geneticvariantsofα1AThavebeenidentifiedwiththeZ-typebeingresponsible
forthemostcommonandsevereformofthediseaseinhomozygouspatients[8].Thepoint
mutationE342KinZ-α1ATrenderstheanti-proteasepronetoaggregationandunabletobese-
cretedintothebloodstreamresultingina90%decreaseinNEinhibitionwithinthelungs.Ac-
cumulationofpolymersofZ-α1ATintheendoplasmicreticulum(ER)ofhepatocytesleadsto
proteotoxicstressandassociatedliverdiseases[9–11].Inadditiontosequestrationofpolymers
intheERofhepatocytes,theE342Kmutationhastwoadditionaldisease-causingeffects.It
causesZ-α1ATtobe5-foldlesseffectiveinaccomplishingitsinhibitoryfunction[12,13]andit
promotesthespontaneousformationofZ-α1ATpolymerswithinthelungs,therebyfurtherre-
ducingthealreadydepletedlevelsofα1ATthatareavailableforalveolaprotection.[14]More-
over,theconversionofZ-α1ATfromamonomertoapolymerrendersitachemoattractantfor
humanneutrophils[15,16].Tosummarize,emphysemaassociatedwithZ-α1ATDresultsfrom
acombinationof(1)lossoffunctionoftheanti-protease,whichleadstotheabsenceofcirculat-
ingα1AT,decreaseofitsinhibitoryactivity,andintra-alveolarpolymerization,and(2)gainof
toxicfunctionfromtheneutrophilchemotacticpropertiesofintra-alveolarpolymers.
PreventingformationandaccumulationofZ-α1ATpolymerscouldbecrucialtotreat
α1ATD[17].ForthisreasonthemechanismsbywhichZ-α1ATformpolymershavebeen
underintenseinvestigation.Asthesubstitutionoftheglutamicacidresidueatposition342by
alysineprovokesaperturbationinthenativestructurebyopeningtheβ-sheetA,biochemical
(cid:1)
evidencerevealstheformationofanunstableandpolymerogenicintermediateM withitsown
RCLpartiallyinserted[18].Theopeningofthes4Acavityallowsthecreationofasequentialβ-
strandlinkagebetweentheRCLofoneserpinandβ-sheetAofanother,leadingtotheforma-
tionofadimerandthenpolymers[6,19–21].TwoadditionalmodelsforZ-α1ATpolymeriza-
tionhavebeenrecentlyproposed,basedonX-raycrystallographyexperiments,suggestingthat
assemblypathwaysofZ-α1ATcouldbediverseandthereforearisingfromstructurallyand/or
dynamicallydistinctpolymerogenicintermediates.Thesemodelsarethes4A/s5Amodelob-
tainedfromaGndHCl-induceddimerofarelatedserpin,antithrombin[22],andtheC
term
swapmodelbasedonaheat-inducedtrimerofadisulfidemutantofα1AT[23].However,in
additiontothesealternativemechanismsofpolymerizationobtainedafterGnd-HClorheatin-
duction,itshouldbenotedthatthes4A/s5Apolymersarenotrecognizedbyaconformation-
specificmonoclonalantibodyofpathologicalα1ATpolymer(Ab2C1)[23–25].
VariousstrategieshavebeenpursuedinordertopreventorevenattenuateZ-α1ATpoly-
merizationsuchasincreasingthemutantproteinsecretionwiththeuseofosmolytes[26–28],
PLOSONE|DOI:10.1371/journal.pone.0126256 May11,2015 2/18
Z-α1ATPolymerizationInhibitor
orblockingZ-α1ATpolymerizationbyeitherfillingthes4Acavitywithpeptides[18]or
crowdingthehydrophobicsidepocketofZ-α1ATwithsmallcompoundsscreenedvirtually
[29].Whileextensiveprogresshasbeenmade,noneofthesestrategieshasbeenentirelysuc-
cessfulsofar.Toachievethisgoal,wedevelopedasetofnovelandintegratedinvitroandinsil-
icoscreeningsmethods;theinvitro,beingahigh-throughputscreeningassayusingamodified
smallpeptidepreviouslyreportedasas4Acavityfiller[18],andtheinsilicobeingavirtual
dockingmodelabletopredictandhelprationalizethebindingofcompoundstoα1AT,includ-
inginthes4Acavity.Here,wepresenthowusingthesetwocombinedmethodswewereableto
identify,rationalizeandconfirmS-(4-nitrobenzyl)-6-thioguanosineasaninhibitorofZ-
α1ATpolymerization.
MaterialandMethods
GeneralMaterialsandMethods
Thepeptideacetyl-FLEAIGGG-Q-GKKGcontainingthe6-mersequenceoftheRCLwassyn-
thesizedbycustomsolid-phasefromtheKeckBiotechnologyCenteratYaleUniversity(http://
info.med.yale.edu/wmkeck/).Abiotinylatedversionofthepeptide(bPEG-peptide)wasob-
tainedbyappendingabiotinyl-polyethyleneglycolspacerontheγ-amidegroupoftheglutami-
nylresidue.ThepresenceoftheLysresiduesconferapositivenetchargetothepeptideat
neutralpH,enhancingitsgeneralsolubility.ThewildtypeandZ-α1ATproteins,preparedac-
cordingtopublishedprotocol,[30]weregraciouslyprovidedataconcentrationof1mg/mlby
ProfessorLomas,CambridgeInstituteforMedicalResearch,UniversityofCambridge,UK,
andstoredat4°C.
Therabbitanti-humanα1ATantibody(serumfractionsIgG)waspurchasedfromAbcam,
Cambridge,MA.
ThetestgroupRK-001oftheLOPAClibrary(LibraryofPharmacologicallyActiveCom-
pounds,Sigma-RBI,Natick,MA)containing80lyophilizedchemicalcompoundswasprepared
ina96-wellplateformat.Allcompoundswereresuspendedin2mlDMSOataconcentration
ofapproximately4mM,basedonanestimatedMWaverageof500g/moles,andstoredat4°C.
PreparationofthebPEG-peptide
ThesynthesizedbPEG-peptidewasfirstsolubilizedin50%formicacidataconcentrationof
~1mg/ml,injectedontoaZorbaxC3ColumnandpurifiedbyRP-HPLCatarateof4ml/min.
Theresultingpurifiedpeptidewasthenlyophilized,resuspendedintoH Oandstoredat-20°C.
2
Afteraminoacidanalysisofthepeptide(CommonwealthBiotechnologiesInc.,Richmond,
VA),variousamountswereinjectedontoRP-HPLCinordertoestablishastandardcurve,al-
lowingustodeterminetheexactconcentrationofeachnewbatchofpurifiedpeptidethat
weprepared.
PreparationofWorkingCompoundPlates
LOPACcompoundsweretransferredfromtheiroriginal96-wellplatestonew96-wellworking
plateswithlowevaporationlid(BDFalconplatesnontreated,BectonDickinsonLabware,San
Jose,CA)inrespecttotheirinitiallocation,andadjustedtoaconcentrationof1mMinPBS
1Xcontaining50%DMSO.FirstandlastcolumnswerefilledonlywithPBS1X/DMSO(50/
50).Workingplatesweresealedwithanadhesiveoverlay,coveredandstoredat4°Cuntil
furtherutilization.
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Z-α1ATPolymerizationInhibitor
SetupoftheMicroplateScreeningAssay
TheassayisbasedontheprincipleofacompetitiveELISA[31].Wellswerecoatedbypassive
adsorptionwitha1/1000solutioninPBS1Xofcaptureα1ATAb.Thescreeningmicroplate
wassealedwithanadhesiveoverlayandincubatedfor2hat37°C.Thewellswerethenwashed
threetimeswithextensionbuffer(PBS1Xand0.01%Tween20),blockedfor1hat37°Cwith
0.3%gelatinandwashedagain.Screeningresultsdescribedinthispaperwerecarriedoutwith
screeningmicroplatesfreshlymade.However,screeningmicroplatescanbefilledwithPBS1X,
hermeticallysealedandstoredat4°Cforoneweekpriortouse.
Z-α1ATPolymerizationInhibitionAssay
Inparallelwiththepreparationofthemicroplatescreeningassay,polymerizationreactions
werecarriedoutin96-wellplateswithorwithouttheLOPACsmallcompounds.A100molar
excessofbPEG-peptidewasusedwith4μg/wellofZ-α1AT.
Eachpolymerizationreactionplatewasorganizedasfollows:thefirstcolumncontained
onlybPEG-peptide(backgroundcontrol);thesecondtoelevencolumnscontainedZ-α1AT,
compoundsandbPEG-peptide;andthelastcolumncontainedZ-α1AT,bPEG-peptideandno
compound(reactioncontrol).Assaywellsweresetupbyaddingtoeachwell20μlofprotein
and20μlofcompoundfromaworkingplate.After3min,160μlofbPEG-peptideat48μM
wasadded.Theplatewasthensealed,shakenonamicroplateshakergentlyfor5stoensure
homogeneityofthedifferentreactants,andplacedat37°Cfor16h.Allwellscontained5%
DMSO.
Attheendofthe16hincubationtime,100μlfromeachwellweretransferredintothecor-
respondingwellofthemicroplatescreeningassay.Onehourlater,thescreeningplatewas
washedthreetimesandthenincubatedinthedarkfor1hatroomtemperaturewith100μl/
wellof1ng/μlofeuropiumstreptavidin(PerkinElmer,Boston,MA)in0.5%BSA-extension
buffer.Threefinalwashesinextensionbufferwerecarriedoutandtheeuropiumwasreleased
fromstreptavidinbytheadditionof100μlofenhancementsolution(PerkinElmer).After5
min,europiumfluorescencewasmeasuredbytime-resolvedfluorometryinaVictor2counter
(PerkinElmer)andthenconvertedtofmolesofbPEG-peptidesrecruitedintoZ-α1AT.Assays
wereconductedintriplicatebyprocessingthreeidenticalplatesinparallel.
DeterminingIC ValuesofInhibitors
50
Asdescribedabove,4μg/wellofZ-α1ATwereincubatedfor3minwithvariousconcentrations
ofacompoundidentifiedasaninhibitor,thehighestconcentrationstartingat400μM.The
concentrationsofthecompoundswererevisedaccordingtothetrueMWofthemolecule.Fol-
lowingthe3minincubation,160μlof48μMbPEG-peptidewereaddedandtherestofthe
protocolwasappliedasdescribedabove.
Z-α1ATPolymerizationSedimentationAssay
Asolutionof0.1mg/mlofZ-α1ATinPBS1Xwasincubatedat37°Cwithorwithout100μM
ofS-(4-nitrobenzyl)-6-thioguanosine.Progressofthepolymerizationreactionwasfollowedby
quantitativeRP-HPLConcentrifugationsupernatants(20min,20,000×g)ofreactionaliquots.
QuantitativedeterminationoftheZ-α1ATmonomerdisappearancewascalculatedaccording
toapre-establishedstandardcurve.
PLOSONE|DOI:10.1371/journal.pone.0126256 May11,2015 4/18
Z-α1ATPolymerizationInhibitor
PreparingProteinStructuresforHomologyModelingandDocking
Simulations
ThecrystalstructuresofthemutantZ-α1AT(PDBcode:3T1P)andthewildtypeM-α1AT
(PDBcodes:3CWMand1QLP)wereobtainedfromtheRCSBProteinDatabase[23,32,33].
UsingtheprogramMOE(MolecularOperatingEnvironment)[34],co-crystallizedwatermole-
culesweredeletedfrombothstructures.Forthepolymerizedmutant(3T1P),onlythefirst
monomerwasretainedandthes4Abindingcavitywascreatedbetweenthes3A/s5Abydelet-
ingresidues345–356,whichcorrespondtotheinsertedresiduesoftheRCL.Theprotonation
stateofatomswasassignedusingProtonate3D[35]utilityinMOEatpH7,300Kand0.1M
saltconcentration.Solventeffectswereimplicitlyincludedbyusingadistance-dependentdi-
electricfunction.PartialchargeswereassignedtoreceptoratomsusingMMFF94s[36]force
fieldparametersasimplementedinMOE.
HomologyModelingProcedure
(cid:1)
ModelingoftheM intermediatestatewasperformedusingtheHomologyModelfacilityin
MOEthatallowsthebuildingofamodelutilizingmultipletemplatestructuressimultaneously.
TogeneratetheM(cid:1)intermediatemodel,twodistincttemplatestructuresofα1AT(PDBcodes
1QLPand3T1P)wereused.Thesetwotemplateproteinsareclosematchesofeachotherwith
98.6%sequenceidentity,differingmostnotablybyamutationreplacingGlu342(structure
1QLP)byLys342(structure3T1P),whichdistinguishesthewild-typeproteinfromthepoly-
merizingZmutantvariant.Duetothedynamicnatureofα1AT,thetwoPDBmodelsrepresent
distinctstable-statestructuresoftheprotein.Thewildtype(1QLP)possessesaclosedβ-sheet
AwiththeRCLextendedalongtheouteredgeoftheprotein,followedbytheC loopstably
term
insertedinβ-sheetB;whereas,themutantstructure(3T1P)hasanexpandedβ-sheetAwith
theRCLinsertedandtheC loopofonemonomerextendedinordertodomainswapwith
term
β-sheetBofaneighboringmonomer.Therefore,the1QLPcrystalstructurewasusedasatem-
plateformodelingi)thepositionoftheRCLwhennotinsertedintoβ-sheetAandii)theposi-
tionoftheC loopwithinβ-sheetBwhenitisnotparticipatinginadomainswap.The3T1P
term
mutantstructurewasusedtomodeltheexpandedpositionofβ-sheetAwhileomittings4Ain
ordertoleaveacavitybetweens3A/s5AwheretheRCLwouldotherwisebefound.Fragments
fromeachtemplatestructurewerejoinedattransitionpointsselectedbysuperimposingthe
templatestructuresandchoosingthoseresiduesbetweenfragmentswithnearoverlapping
atompositions.
Sinceproteinsinherentlyhavemanydegreesoffreedom,findingasinglerepresentative
modelisachallengeoftenrequiringsamplingmanypossibleconformationalstates.MOEal-
lowsforsamplinganensembleofpossiblestructuralmodels,notunlikemoleculardynamics,
usingconformationalsamplingtechniquesalongwithhierarchicalroundsofgeometricanden-
ergeticmodelrefinementuntilastablelowenergymodelisidentifiedtorepresenttheensemble
(cid:1)
ofstructures.InthecaseofsamplingpossibleM intermediatestatemodels,atotalof25initial
modelswerefirstgeneratedeachwithuniquecarbonbackbonepositions.Foreachofthose25
models,5additionalmodels(i.e.atotalof125models)werecreatedwithalternatesidechain
positionsinordertoprovidesamplingofpossiblesidechaindegreesoffreedom.Theseinitial
modelswerethenenergyminimizedtoagradientof0.1kcal/mol(cid:3)Å.AfinalM(cid:1)model(Model
126)wascreatedusingtheensembleofstructuresandtheGeneralizedBorn/VolumeIntegral
(GB/VI)energyscoringmethod[37]toselectthebestinitiallypackedstructureandthenthe
structureswerefurtherenergyminimizedtoagradientof0.01kcal/mol(cid:3)Å.
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Z-α1ATPolymerizationInhibitor
DockingSimulationProcedure
Three-dimensionalstructuresofthe80invitrotestedchemicals,includingS-(4-nitrobenzyl)-
6-thioguanosine,wereobtainedinSDFformatfromtheelectronicLOPAClibrary,testgroup
RK-001.TheMMFF94sforcefieldwasusedtoassignparametersandpartialatomicchargesto
eachligand.Energyminimizationofthestructureswasperformedforeachligandtoagradient
of0.1kcal/mol(cid:3)Å.Theenergy-minimizedligandsweredockedintothreestructuralvariations
ofα1AT:M(cid:1)intermediate(HomologyModel126),mutantZ-α1AT(PDB:3T1P)andwild
typeM-α1AT(PDB:3CWM)usingthedockingfacilitybuiltintoMOE.UsingtheMOESite
Finderfacility,severallikelybindingsiteswereidentifiedforeachoftherespectivereceptor
(cid:1)
models(wildtype,mutant,andM intermediate).Thistoolusespolarandnonpolarspheres
(1.4Åand1.8Åinradius,respectively),tosampletheproteinsurfacefornonpolarorpolar
contactpointsandsaveslocationsoftheproteinas‘bindingsites’iftheycontainthreeormore
adjacentspheresmakingfavorablecontactwiththeproteinsurface.Thesitesidentifiedfor
eachreceptortypearelistedinTable1.Dockingcalculationsofthe80compoundswerecarried
outseparatelyforeachofthethreereceptorstructuresandateachpotentialbindingsite.Dur-
ingdocking,initialplacementofligandatomsineachpotentialbindingsiteusedtheTriangle
Placementmethod.ThebindingfreeenergyofeachposeusingtheLondondG[34]scoring
method.ThetopfivescoringposeswerefurtherenergyminimizedusingtheMMFF94sforce
field,allowingligandatomsandproteinsidechainswithin6Åofeachdockedligandtobe
treatedasflexible.Atetheringweightof10kcal/mol/Å2wasappliedtopartiallyrestrainflexible
Table1. DockingresultsfromM-α1AT,Z-α1AT,andM*modelwithS-(4-nitrobenzyl)-6-thioguanosineandthe79othersmallmolecules.
Structure SITE LowestEnergyβ(kcal/mol) B9Energyδ(kcal/mol) B9Rankγ
M-α1AT(3CWM) SITE1 n/aΘ n/a n/a
SITE2 -7.0 -5.6 19th
SITE3 -6.2 -5.3 8th
SITE4 -6.4 -3.4 25th
SITE5 n/a n/a n/a
SITE6 -7.6 -7.6 1st
Z-α1AT(3T1P) SITE1 n/a n/a n/a
SITE2 -6.5 -3.9 34th
SITE3 -9.0 -5.8 13th
SITE4 -4.6 -3.6 19th
SITE5 n/a n/a n/a
SITE6 n/a n/a n/a
M*Model SITE1 -10.7 -7.9 7th
SITE2 -10.7 -8.8 5th
SITE3 -5.1 -4.6 9th
SITE4 n/a n/a n/a
SITE5 -8.4 -8.4 1st
SITE6 -6.2 -4.22 12th
αSitenumberwhereS-(4-nitrobenzyl)-6-thioguanosine(B9)wasdocked.
βLowestobservedbindingenergy(kcal/mol)foranyofthe80dockedcompounds.
δPredictedbindingenergy(kcal/mol)forB9.
γRankofB9relativetothebindingenergiesforall80dockedcompounds.
ΘSitenumbersthatarenotfoundinagivenmodelarenotedbyanotapplicablesymbol(n/a).
doi:10.1371/journal.pone.0126256.t001
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Z-α1ATPolymerizationInhibitor
atomsaroundtheiroriginallocation.Afinaldockingscore/bindingfreeenergyestimatefor
eachenergy-minimizedposewascalculatedusingtheAffinitydG[34]scoringmethod.
ResultsandDiscussion
CharacteristicsoftheZ-α1ATPolymerizationInhibitorScreeningAssay
Previousstudieshaveshownthata6-merpeptidewhoseaminoacidsequencecontainsthe
RCLsequenceFLEAIGcanspecificallybindtheZ-mutantatitsopeneds4Apocket,butnot
thewildtype[18].TheZ-α1AThigh-throughputmicroplatescreeningassayisbasedonthis
concept.Thus,wedesignedasimilarpeptideandaddedabiotin-polyethyleneglycol(bPEG)
tagattheC ofthereactiveloopsequenceaswellassomehydrophilicaminoacidstoin-
term
creasepeptidesolubility.TheinsertionofaPEG-basedspacerpreventspossiblesterichin-
drancebetweenthepeptideandthebiotinmolecule,resultinginbetteravidinbindingand
therefore,amoreaccuratemeasurementofthebiologicalactivity.
ToassesstheabilityofsmallmoleculestoinhibittherecruitmentofthebPEG-peptideinto
Z-α1AT,Z-α1ATissubjectedfor3mintolibrarycompoundbeforeadditionofbPEG-peptide
andtheresultingcomplexformedistransferredintomicrotiterwellscontainingtheattached
antibody.TheamountofbPEG-peptidesincorporatedintothemutantproteinisthendeter-
minedbyaeuropium-streptavidintreatmentandtime-resolvedfluorescencemeasurements.
Theinhibitioneffectofacompoundiscalculatedasapercentagewithrespecttoareaction
control—i.e.Z-α1ATthathasonlybeenexposedtothebiotinylatedpeptideandnottoacom-
pound.Anycompoundshowinganinhibitoryeffectofatleast50%isconsideredasahit.
RegardingitsabilitytobindZ-α1AT,wefoundthebPEG-peptideassociationkineticstobe
infavorofthemutantproteinasewithaninitialassociationrateof0.22±0.08fmoles(cid:3)h-1vs.
0.042±0.1fmoles(cid:3)h-1forthewildtype(Fig1A).Wealsofoundthatanincubationperiodof16
hrsforthepeptidewithZ-α1ATisanadequatescreeningend-pointforthescreeningassayas
thistimeperiodisassociatedwithahighsignal-to-noiseratio.Inaddition,thepresenceof5%
DMSOinthewellsdoesnotaffectthebPEG-peptidebindingkinetics(Fig1B).Sincecom-
poundlibrariesaregenerallystoredinDMSO,thisfeaturemakestheassaywellsuitedfora
high-throughputscreeningassay.
Finally,thisscreeningassayexhibitsverygoodreproducibilityasreflectedbytheerrorbars
showninFig2.ItrequiresonlysmallamountofproteinandlowconcentrationsofbPEG-pep-
tide,whichmakeitbotheconomicalandphysiological.
S-(4-nitrobenzyl)-6-thioguanosineIdentifiedasInhibitorofZ-α1AT
Polymerization
ThetestgroupRK-001(80compounds)ofthesmallcommerciallyavailableLOPAClibrary
containingdrug-likemoleculeswasusedtotesttheperformanceofthescreeningassay.Fig2
showsatypicalscreeningresult.Asindicatedinthefigure,onlyonecompoundofthetested
compoundplateappearsasahit,exhibitinga67±2%inhibitionactivityat100μM.Thiscom-
poundisS-(4-nitrobenzyl)-6-thioguanosine.ToconfirmitsabilitytoinactivateZ-α1ATpoly-
merization,dose-responsescurveswerecarriedoutandanIC of73±0.12μMcalculated(Fig
50
3A).TheIC valueobtainedisinthemicromolarrangeandmatcheswellwiththe
50
screeningresults.
Todefineabetterpharmacophore,andthereforetoidentifyanyadditionalstructuralele-
mentrequiredforinhibitingZ-α1ATpolymerization,wethencomparedourcompoundtothe
entiredatabasethatregroupsalloftheLOPACmolecules.Surprisingly,wefoundanothercom-
poundthatpossessesaverysimilarstructure,differingbyasingleaminogroup,butthatdid
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Z-α1ATPolymerizationInhibitor
Fig1.KineticdiagramofbPEG-peptidebindingtoα1AT.(A)Fourmicrogramsperwellofattached(&)Z-
α1ATor(●)M-α1ATwereincubatedforvarioustimesinpresenceof38.4μMbPEG-peptide.(B)Z-α1ATwas
incubatedinpresenceof5%DMSOandbPEG-peptidefor16h.Errorsbarsreflectthestandarddeviationof
threereplicates.
doi:10.1371/journal.pone.0126256.g001
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Z-α1ATPolymerizationInhibitor
Fig2.Patternofinhibitionresultingfromthescreeningof80unknownLOPACcompounds.A96-well
platewascoatedwith4μg/wellofZ-α1ATandincubatedfor16hwith100μMofvariouscompoundsand
38.4μMofbPEG-peptide.TheblackarrowindicatesthecompoundthatcorrespondstoS-(4-nitrobenzyl)-
6-thioguanosineandgivesaninhibitioneffectof67±2%and.Theerrorbarsarethestandarddeviationof
threeindividualexperiments.
doi:10.1371/journal.pone.0126256.g002
notshowanyinhibitoryeffect,neitherduringtheoriginalscreeningnorinthevalidationassay
(Fig3Aand3B).
ValidationoftheActionofS-(4-nitrobenzyl)-6-thioguanosine
Apolymerizationreactionwassetupinpresenceorabsenceof100μMofS-(4-nitrobenzyl)-
6-thioguanosineandthedisappearanceoftheZ-α1ATmonomermonitoredbyRP-HPLC—a
diminutioninmonomerconcentrationindicatesthattheproteinhasbeenrecruitedintopoly-
mers.AnalysisconfirmedthatZ-α1AThasitspolymerizationratedecreased33timesinpres-
enceofthecompoundandthatitseffectislonglasting(Fig4).
However,asthes4Acavitydoesnotexistinanycrystalstructureofα1AT,atheoretical
(cid:1) (cid:1)
modelcomparabletoM hadtobecreated.TheM intermediatestateisdescribedtohavethe
followingthreestructuralfeatures:i)anexpandedβ-sheetAwithas4Acavitybetweens3A/
s5A,ii)anRCLattheprecipiceofinsertingbetweens3A/s5A,andiii)theC loopinserted
term
withinβ-sheetBandnotparticipatinginadomainswapwithanotherprotein.Theseimpor-
(cid:1)
tantfeaturesoftheM modelarerepresentedinthehomologymodelbuiltfromthetwoavail-
ablecrystalstructuresofα1AT(Fig5).
(cid:1)
TobuildtheM statehomologymodel,atotaloffiveproteinfragmentsofthetwocrystal
structures,1QLPand3T1P,weremerged.Fig6showsthatfragment1consistsofresidues
1–105(1QLP)whichmodeltherightsideofβ-sheetB,withrespecttobetastrandsadjacentto
therightsideoftheC loop.Fragment3consistsofresidues205–291whichconstitutethe
term
leftsideofβ-sheetB,withrespecttobetastrandsadjacenttotheleftsideoftheC loop.To-
term
getherthesetwofragmentsmodelthepositionofβ-sheetBsothattheRCLresiduesfromfrag-
ment5(residues345–394from1QLP)canbeplacedontheoutsideofthes4Apocketalong
withtheC loopburiedwithinβ-sheetB.Thepositionofstrandss1A,s2A,ands3Ainβ-
term
sheetAaremodeledfromfragment2(residues106–204from3T1P),andfragment4(residues
292–344from3T1P)modelsthepositionofstrandss5Aands6A.Together,thepositionsof
fragments2,4and5createthecavitys4Abetweens3A/s5A,whichwouldotherwisebethesite
ofRCLinsertion.Thisopenedconformationofα1ATrepresentsoneofthepossiblestructures
PLOSONE|DOI:10.1371/journal.pone.0126256 May11,2015 9/18
Z-α1ATPolymerizationInhibitor
Fig3.S-(4-nitrobenzyl)-6-thioguanosineinhibitsbPEG-peptidebindingtoZ-α1AT.(A)Dose-response
curveswereassayedforvariousconcentrationsof(●)S-(4-nitrobenzyl)-6-thioguanosineand(&)its
homologueS-(4-nitrobenzyl)-6-thioinosine.(B)Chemicalstructuresof(left)S-(4-nitrobenzyl)-
6-thioguanosineand(right)S-(4-nitrobenzyl)-6-thioinosine.Theerrorsbarsarethestandarddeviationofan
experimentconductedintriplicate.
doi:10.1371/journal.pone.0126256.g003
(cid:1)
oftheunstableM intermediatestateforwhichexperimentalmethodssuchascrystallography
cannotreproduce.
Analysisofα1ATStructuresandtheirPotentialBindingSites
Allofthe80invitroscreenedcompounds,includingS-(4-nitrobenzyl)-6-thioguanosine,were
dockedintoeverypotentialbindingsitetoassessifthecomputationalresultiscomparableto
theinvitroscreening.AbindingsiteabletodockS-(4-nitrobenzyl)-6-thioguanosinewitha
lowerbindingenergythanthe79othercompoundswouldbeapromisingsiteforfurther
experimentalinvestigations.
Sixputativebindingsiteswerepredictedamongthethreeavailableproteinmodels:M-
α1AT,Z-α1ATandintermediateM(cid:1)(Fig7).SITE1andSITE5werebothexclusivelyavailable
PLOSONE|DOI:10.1371/journal.pone.0126256 May11,2015 10/18