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A general method for discovering inhibitors of protein-DNA interactions using photonic crystal biosensors

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Protein-DNA interactions are essential for fundamental cellular processes such as transcription, DNA damage repair, and apoptosis. As such, small molecule disruptors of these interactions could be powerful tools for investigation of these biological
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   AGeneralMethodforDiscoveringInhibitorsof Protein  DNAInteractionsUsingPhotonicCrystalBiosensors LeoL.Chan † ,MariaPineda ‡ ,JamesT.Heeres § ,PaulJ.Hergenrother  §,¶, *,andBrianT.Cunningham †, * † Department of Electrical and Computer Engineering,  ‡ Department of Bioengineering,  § Department of Biochemistry, and ¶ Department of Chemistry, University of Illinois at Urbana  Champaign, Urbana, Illinois 61801 H igh-throughputscreening(HTS)ofcompoundcollectionsisnowastapleofmoderndrugdis-covery.Inthemostcommonincarnation, invitro enzymeinhibitionscreensoflarge(   100,000mem-bers)compoundlibrariesareconductedusingsub-stratesthatprovideaneasilyquantifiedchromogenic/fluorescentreadout.Suchscreenshaveledtothediscoveryofmanynovelenzymeinhibitorsanddrug leads(  1,2  ).Unfortunately,manypotentialdrugtargetsarenotenzymes,andthusforthesesystemshigh-throughputmethodsareneededthatgobeyonden-zymeinhibitionassaysanddirectlyreportonsmallmolecule  proteinbindingevents.Oneareainwhichsmallmoleculeligandsfornonen-zymeproteinswouldbeusefulisinthedisruptionof protein  macromoleculeinteractions.Theidentificationofcompoundsthatperturbprotein  proteinor protein  nucleicacidinteractionsisextremelychalleng-ing(  3–6  ),andthisispartlyduetothepaucityofgoodhigh-throughputscreens.Successesinmodulating protein  proteinandprotein  nucleicacidinteractionswithsmallmoleculesfallintoafewclasses:surfacereceptor   ligandinteractions(integrins(  7–9  ),IL-1/2(  10,11  ),TNF   (  12  )),cytoplasmictargets(iNOS(  13  ),HIVprotease(  14  ),Bcl-2/xL(  15  ),XIAP(  16  )),andtranscription-relatedtargets(  17   ).Thislastclassofcom-poundscanelicittheiractionbyinhibitingtranscriptionfactordimerization/DNA-binding(B-ZIP(  18  ),Zn-finger proteins(  19  ),STAT3(  20,21  ),c-Myc/Max(  22,23  ),HIF-1(  24  ),GLI(  25  )),byrelievinginhibitoryproteins(p53/MDM2(  26  )),orbyrecruitingtranscriptionfactorsorco-activatorstocertainDNAsequencesbyusingpoly-amides(  27,28  )orsmallmolecules(  29–31  ).Inorder tofurtherfacilitatetheidentificationofinhibitorsof  *Corresponding authors,bcunning@uiuc.edu,hergenro@uiuc.edu. Received for review March 13, 2008and accepted May 22, 2008.Published online June 27, 200810.1021/cb800057j CCC: $40.75 © 2008AmericanChemicalSociety  ABSTRACT   Protein  DNAinteractionsareessentialforfundamentalcellularpro-cesses such as transcription, DNA damage repair, and apoptosis. As such, smallmolecule disruptors of these interactions could be powerful tools for investigationof these biological processes, and such compounds would have great potentialas therapeutics. Unfortunately, there are few methods available for the rapid iden-tification of compounds that disrupt protein  DNA interactions. Here we showthat photonic crystal (PC) technology can be utilized to detect protein  DNA inter-actions, and can be used in a high-throughput screening mode to identify com-pounds that prevent protein  DNA binding. The PC technology is used to detectbindingbetweenprotein  DNAinteractionsthatareDNA-sequence-dependent(thebacterial toxin  antitoxin system MazEF) and those that are DNA-sequence-independent (the human apoptosis inducing factor (AIF)). The PC technology wasfurther utilized in a screen for inhibitors of the AIF   DNA interaction, and throughthisscreenaurintricarboxylicacidwasidentifiedasthefirst in vitro inhibitorofAIF.The generality and simplicity of the photonic crystal method should enable thistechnology to find broad utility for identification of compounds that inhibitprotein  DNA binding. A RTICLE www.acschemicalbiology.org  VOL.3 NO.7 •  ACS CHEMICAL BIOLOGY  437  protein  nucleicacidinteractions,wesoughttode-velopahigh-throughputscreeningmethodthatwoulddirectlyreportontheinhibitionofprotein  nucleicacidcomplexes.Thereareseveraltechniquesutilizedtomeasureprotein  nucleicacidbinding.CurrentmethodologiesincludeDNAmicroarrays(  32  ),fluorescenceanisotropy(  33  ),electrophoreticmobilityshiftassays(EMSA)(  34  ),DNA/RNAfootprinting(  35,36  ),chromatinimmunopre-cipitation(ChIP)(  37   ),isothermaltitrationcalorimetry(ITC)(  38  ),andsurfaceplasmonresonance(SPR)(  38,39  );eachisabletomeasurethedegreeand/orspecific-ityofprotein  DNA/RNAbinding.Whiletheaforemen-tionedtechniquesareusefulindeterminingbindingaf-finities,fewareoptimalforHTSanddrugdiscovery.DNAmicroarraysandemergingSPRmicroscopytechniquesmayultimatelybesuitableforHTS,andthetechnologyreportedhereinmaybeviewedascomplementarytotheseassays.However,DNAmicroarrayandSPRmicros-copybasedscreeninghasgenerallybeenappliedtotheidentificationofoptimalpromoterbindingsitesfor transcriptionfactors(  33,40–42  )andnotindrugdiscov-ery.Fluorescenceanisotropy(alsotermedfluorescencepolarization,orFP)hasbeenwidelyusedforthemea-surementofprotein  proteinandprotein  nucleicacidinteractions(  33  ),aswellasthescreeningofcompoundlibrariesforinhibitorsoftheseinteractions(  43–45  ).Al-thoughfluorescenceanisotropyisapopularmethodfor suchexperiments,therearesomelimitationsonfluores-cenceanisotropyasaHTSmethod.Onelimitationistherecommended  10-foldmassexcessofthenonflu-orescentbindingpartner(  46  ),althoughthereareex-amplesshowingthatfluorescenceanisotropycanbeeffectivebelowthislimit(  47   ).Anotherlimitationisthepotentialforfalsepositivesduetofluorescentcompounds, which is an inherent limitation of anyfluorescence-basedHTSmethod.Giventhelargelyunex-ploredpharmacologicalrealmthatisprotein  nucleicacid interactions, HTS assays independent of fluores-centtagswouldbeextremelyuseful,especiallyinthosecaseswherefluorescenceanisotropyisnotpossible.Inthisreportwedescribethefirstuseofphotoniccrystaltechnologyforthedevelopmentofanassaycapableof detectingprotein  DNAbindingandfurtherapplyitinahigh-throughputscreeningmodefordiscoveryofinhibi-torsofaprotein  DNAinteraction. RESULTSANDDISCUSSION Asstatedabove,fluorescencepolarizationhasbeensuccessfullyutilizedinmultiplehigh-throughputscreens,includingsomewhosegoalwastheidentifica-tionofinhibitorsofprotein  DNAinteractions(  18,23  ).Thus,inoursearchforinhibitorsoftheapoptosisinduc-ingfactor(AIF)  DNAinteraction,weinitiallyattemptedtodevelopanFP-basedHTS.Unfortunately,DNAse-quenceswithtwodifferentfluorescenttagsdidnotgiveanoticeablechangeinfluorescencepolarizationuponincubationwithAIF(seeSupplementaryFigure1).WeascribedthefailureoftheFPmethodinthiscasetothelowaffinityofAIFforanyDNAsequence,asdemon-stratedbythemolarratiosrequiredinEMSA(  48,49  ).Inaddition,theFPassaywithAIFiscomplicatedbythefact that AIF itself is fluorescent due to the presence of itsflavincofactor.Wethussoughttodevelopageneraland high-throughput technique for the identification of inhibitorsofprotein  DNAinteractionsthatwouldbeabletoavoidthesecomplications.Anewclassofdisposablemicroplate-basedopticalbiosensorsbasedontheuniquepropertiesofphotoniccrystals(PC)havebeenrecentlydevelopedbyCunning-ham  et al . (  50  ). Like other optical biosensors, including thoseutilizedinSPR,PCbiosensorsdetectbiomolecular interactionsonthesurfaceofatransducerthroughchangesindielectricpermittivitywithrespecttotheliq-uidmedia.APCiscomposedofaperiodicarrangementofdielectricmaterialthateffectivelypreventspropaga-tionoflightatspecificwavelengthsanddirections.Whenilluminatedwithwhitelight,appropriatelyconfig-uredphotoniccrystalsareabletoreflectnarrowbandlightwhosewavelengthisdirectlydependentonthelo-caldensityofadsorbedbiomolecules(Figure1).Asso-ciationofmacromoleculestothesensorsurfacemodu-lates the peak wavelength value (PWV) of the reflectedlight,allowingfordetectionofbindingbyashiftinthePWV.Photoniccrystalbiosensorsincorporatedontostandardformat96-,384-,or1536-wellmicroplateshavebeenusedtodetectantibody  antigen,smallmolecule  protein,andwholecellinteractionsonthebiosensorsurfacewithouttheuseoffluorescentlabels(  50  ).Intheworkdescribedherein,photoniccrystaltech-nologyisappliedtothedetectionandanalysisof protein  DNAinteractions.Todemonstratethescopeofthismethod,wechosetwoverydifferentprotein  DNAinteractions:thebacterialMazEFcomplex,whichbindstoitspromoterDNAinasequence-specificman- 438  VOL.3 NO.7 • 437–448 • 2008  www.acschemicalbiology.org CHAN  ET AL.  ner,andthehumanAIF,aproteinthatbindsnonspecif-icallytochromosomalDNA.MazEFisabacterialtoxin  antitoxinsystemthoughttoberesponsibleforthemaintenanceofresistance-encodingplasmidsincertaininfectiousbacteria(  51–53  ).Originallyidentifiedonthe E.coli  chromosome,MazEFisaheterohexameric,  77kDacomplexconsist-ingof1MazE(antitoxin)dimer,andtwoMazF(toxin)dimers(  54  ).MazFisanRNasewhichisreleasedfromMazEuponplasmidloss,resultingininhibitionofbacte- Photonic crystal biosensor Spectrometer  Wavelength (nm)    R  e   l  e  c   t  a  n  c  e Reflected light Incident lightCollimating lensOptical fiber probeBroadband light emitting diode( λ  = 840 − 890 nm)Detecting fiber Illuminating fiber  a bc = Streptavidin= Biotinylated DNA= Blocking agent= DNA binding protein Wavelength (nm)840 890880870860850 0.70.00.10.20.60.50.40.3    R  e   f   l  e  c   t  e   d   i  n   t  e  n  s   i   t  y   (  a  r   b   i   t  r  a  r  y  u  n   i   t  s   ) Wavelength (nm)840 890880870860850 0.70.00.10.20.60.50.40.3    R  e   f   l  e  c   t  e   d   i  n   t  e  n  s   i   t  y   (  a  r   b   i   t  r  a  r  y  u  n   i   t  s   ) Figure 1. a) Schematic of the PC biosensor. A broadband LED illuminates the biosensor from the bottom, and reflected light is collected and trans-ferred to a spectrometer where the PWV is measured. b) Image of PC biosensor films adhered to the bottom of black 384-well plates. c) Diagram of protein  DNA binding experiments performed with PC biosensors. Streptavidin-coated biosensors are used to bind biotinylated DNA oligomers, anda distinct peak wavelength of the reflected light is observed. After the addition of Starting Block (Pierce Biotechnologies), a DNA-binding protein isadded, and a shift in the wavelength of reflected light is observed. A RTICLE www.acschemicalbiology.org  VOL.3 NO.7 • 437–448 • 2008  439  rialgrowth(  55  ).Inadditiontoitstoxicaction,theMazEF complexalsoregulatesitsexpressionbybindingtoitsownpromotersequence(  55  ).AIF is a mammalian mitochondrial NADH-oxidoreductasethatalsohasakeyroleincaspase-independentcelldeath(  56,57   ).The67kDaformof AIFisproducedinthecytoplasm,whereitthentranslo-catestothemitochondriaandcarriesoutitsoxidoreduc-tasefunctionaswellaspossibleupkeepofcomplexI(  58  ).Uponcellularinsults(suchasDNAdamage),AIFiscleavedofftheinnermitochondrialmembraneandre-leasedintothecytoplasmasa57kDaprotein.Onceinthecytoplasm,AIFtranslocatestothenucleusandbindstoDNAinasequence-independentfashion,causing stageIchromatincondensation,eventuallyleadingtocelldeath.AIFisthoughttocontactDNAthroughelectro-staticinteractions,asmutationsofsurfacelysineandarginineresiduesabrogateDNAbinding  invitro andincell culture (  49  ). These surface residues are containedwithin the FAD-binding domain and the C-terminal do-mainofAIF,anditisproposedthat12basepairsof double-stranded DNA (dsDNA) can be bound in thisstretchofAIF(  49  ).AlthoughthecrystalstructureofAIF hasbeensolved(  49,59  ),nococrystalstructurehasbeenobtainedforAIFandDNA.Smallmoleculeinhibi-torsoftheAIF   DNAinteractionareofgreatinterestduetotheinvolvementofAIFinmultiplediseasestatemod-elsincludingParkinson’sdisease(  60  ),ischemia/reper-fusioninjury(  61  ),amyotrophiclateralsclerosis(  62  ),andHuntington’sdisease(  63  );however,nosmallmoleculeinhibitorsoftheAIF   DNAinteractionhavebeenreported. SensorFabrication. ThePCopticalbiosensorsusedinthisworkhavebeendescribedpreviously(  50  ).Briefly,thesensorcontainsaone-dimensionalsurfacegrating structurewithaperiodof550nm(Figure1,panela).Itisproduced via aroom-temperaturereplicamoldingpro-cessusingaUV-curablepolymeronatransparentpoly-estersheet.Thelowrefractiveindexpolymergrating structureissubsequentlycoatedwithafilmofhighre-fractiveindexTiO 2  toachievethefinalsensorstructure.Thecompletedsensoriscutfromthepolyestersheetandattachedtothebottomofastandard384-wellmi-croplate(Figure1,panelb).Thereadoutinstrument(SRUBiosystemsBINDReader)(  50,64,65  )illuminatesmicroplatewellsfrombelowwithabroadbandlightsourcecoupledtoeightopticalfibers,eachilluminat-inga  2mmdiameterregionofthePCsurfaceatnor-malincidence.Reflectedlightiscollectedbyasecondopticalfiber,bundlednexttotheilluminatingfiber,andmeasuredbyaspectrometer.Anautomatedmotionstageenablesparallelcollectionofreflectancedataattimedintervalstoacquirekineticinformationfromall384wells.InFigure1,panelcillustratesthegeneralex-perimentalsetupofDNA-bindingassaysperformedus-ingPCbiosensors. Sequence-DependentDNABinding:MazEF. MazEF wasshownpreviouslytobindtoitsownpromoterse-quenceusinganelectrophoreticmobilityshiftassay(EMSA)(  55  ).MazEhassomeintrinsicDNAbindingabil-ity,whileformationoftheMazEFcomplexdramaticallyincreasesDNAbinding(  55  ).Fortheexperimentsde-scribedherein,thesamepromotersequenceusedbyZhangandco-workers(  55  )waspurchasedwithoneendfunctionalizedwithbiotin.Thesensorsurfacewascoatedwith12  Lof1  MbiotinylatedDNA(12hat4°C)andblockedwithStartingBlock(PierceBiotechnol-ogy)for2hat4°C.MazEFwasexpressedandpurifiedasdescribed(  76  )andthenaddedtoDNA-containing wellsatthespecifiedconcentration(Figure2,panela)for1hat25°C.InFigure2,panelashowstheassocia-tionoftheMazEFproteincomplexwithbiosensorscoatedwithpromoterDNA.Thisassociationwasinhib-itedbypreincubatingMazEFwithincreasingconcentra-tionsoffreepromoterDNAfor15min(Figure2,panelb).ThebindingofMazEFtoitspromotersequencewasalsospecific;whenacontrolsequenceofGC-richDNAwithoneendbiotinylatedwascomplexedtothePCbio-sensor,MazEFexhibitedonlyminimalbinding(Figure2,panelc).ThekineticsofMazEFbindingtothepromoter-boundbiosensorwerealsomonitoredoverthecourseof 30min(Figure2,panelc). Sequence-IndependentDNABinding:AIF. AnalysesoftheDNAbindingpropertiesofthe57kDaformofAIF (AIF   1-121)havebeenperformedpreviouslyusing EMSA(  48  ).AIFbindsDNAnonspecifically,asdifferentsequencesoffreeoligomerareabletopreventAIFbind-ingtoaDNAladder(  48  ).Therefore,abiotinylated,ran-domized30bpsequenceofdsDNAwaschosenastheDNAtargetofAIF;ithasbeenshownthatAIFiscapableofbindingDNAofthislength(  48  ).PreparationofthebiosensorsurfacewasanalogoustotheMazEFexperi-mentsdescribedabove,exceptthespecifiedconcentra-tionsofAIFwereincubatedwithbiotinylatedDNAfor 30minat25°C.AsmonitoredbythePCbiosensor,theassociationofAIFwithbiotinylatedDNAwasfoundtobe 440  VOL.3 NO.7 • 437–448 • 2008  www.acschemicalbiology.org CHAN  ET AL.  pHdependent(Figure3,panela),andapHof 6.3wasfoundtogivemodestPWVshiftswhilemaintainingproteinstability.ThispHdepen-denceisnotsurprisingduetothefactthatthepIofAIF   1-121is7.8,andapHlowerthanthepIwouldfavorbindingtoaDNAsubstrate.InFigure3,panelbshowstheassociationofAIF withbiotinylatedDNA;thisinteractionisalsoin-hibitedbya15minpreincubationwithfreeDNA(Figure3,panelc).AIFisthoughttobindDNAinacooperativefashion,duetothefactthatalargemolarexcessofAIFisrequiredtodetectbinding (  48  ).BecauseMazEFbindstoitspromoterse-quencespecifically,andnoknowncooperativeinteractionhasbeenpostulated,weproposethedifferenceinPWVshiftvaluesbetweenMazEF andAIFarebecauseofthedifferenceintherela-tiveaffinitiesoftheseproteinsfortheirDNAtargets. DemonstrationofHTSPotential:Screening forInhibitorsoftheAIF   DNAInteraction. ThedatainFigures2and3demonstratethatthePCbiosensorcanbesuccessfullyusedtodetectprotein  DNAinteractions.Withtheseexperi-ments in place, we moved to develop a high-throughputscreenthatcouldbeusedtoiden-tifycompoundsthatpreventtheAIF   DNAinteraction.Aswithpreviousexperiments,a1  MsolutionofbiotinylatedDNAwasimmobi-lizedonstreptavidin-coatedPCbiosensors,andStartingBlockwasthenaddedtoreducenon-specificinteractionsbetweenAIFandthebiosen-sorsurface.AIF(3.51  M)andputativesmallmoleculeinhibitors(25  M)wereincubatedto-getherfor15minat25°Cinaclear384-wellplate(Falcon);referencewellsforeachcom-poundwerealsopreparedinthesame384-wellplate.ThesesolutionswerethentransferredtotheDNA-containing384-wellbiosensorplate.CompoundsthatinhibittheAIF   DNAinterac-tionwouldpreventthePWVshiftobservedintheAIF   DNAbindingevent.Inthisfashion,approxi-mately1000compounds(obtainedfromanin-housecompoundcollection(  66  ))werescreenedinduplicateataconcentrationof25  M.Allex-perimentalwellswerenormalizedagainstthefollowingtworeferencewells:AIFwithnobiotin-ylated DNA (to account for the nonspecific inter- abc 0.05.04.54.03.53.02.52.01.51.00.5    P   W   V  s   h   i   f   t   (  n  m   ) 0 26.513.26.63.31.70.80.40.20.1 [MazEF] ( µ M)0.01.21.00.80.60.40.2    P   W   V  s   h   i   f   t   (  n  m   ) 0 12.56.23.11.60.80.40.20.10.05 [Free promoter DNA] ( µ M)25Promoter ControlNo DNA0.03.53.02.52.01.51.00.5    P   W   V  s   h   i   f   t   (  n  m   ) − 0.50 12012 21 30 39 48 57 66 75 84 93 102 111Time (min) Figure 2. a) MazEF associates with its promoter sequence bound tothe PC biosensor surface in a dose-dependent fashion. b) Prein-cubation of MazEF (1.8  M) with its nonbiotinylated promoter se-quence reduces the association of MazEF with the promoter-boundbiosensor surface. c) Kinetics of MazEF (0.2 mg mL -1  ) binding toits own promoter sequence. A rapid increase in PWV shift is ob-served upon MazEF addition to the promoter-bound biosensor sur-face. In contrast, MazEF showed little affinity for a biotinylatedalternating GC control DNA of the same length as its promoter se-quence, similar to its association blocked sensor surface (no DNA). All error bars represent the calculated standard error (  n  3). A RTICLE www.acschemicalbiology.org  VOL.3 NO.7 • 437–448 • 2008  441
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