by! Kristen!S.A.!Sheffield! A!thesis!submitted!in!partial!fulfillment! of!the!requirements!for!the!degree!of! Master!of!Science!(MSc)!in!Biology!

Transcription

1 TheidentificationandcharacterizationofsmallmoleculesfortheinhibitionofNGF andprongf8p75 NTR interactionsusingsurfaceplasmonresonancespectroscopy by KristenS.A.Sheffield Athesissubmittedinpartialfulfillment oftherequirementsforthedegreeof MasterofScience(MSc)inBiology TheFacultyofGraduateStudies LaurentianUniversity Sudbury,Ontario,Canada KristenS.A.Sheffield,2015

2 THESIS DEFENCE COMMITTEE/COMITÉ DE SOUTENANCE DE THÈSE Laurentian Université/Université Laurentienne Faculty of Graduate Studies/Faculté des études supérieures Title of Thesis Titre de la thèse The identification and characterization of small molecules for the inhibition of NGF and prongf-p75 NTR interactions using surface plasmon resonance spectroscopy Name of Candidate Nom du candidat Degree Diplôme Sheffield, Kristen Master Science Department/Program Date of Defence Département/Programme Biology Date de la soutenance March 11, 2016 Thesis Examiners/Examinateurs de thèse: Dr. Gregory Ross (Supervisor/Directeur(trice) de thèse) Dr. Ashley Scott (Co-supervisor/Co-directeur(trice) de thèse) Dr. Tom Kovala (Committee member/membre du comité) APPROVED/APPROUVÉ Dr. Michael Kawaja (External Examiner/Examinateur externe) Approved for the Faculty of Graduate Studies Approuvé pour la Faculté des études supérieures Dr. David Lesbarrères Monsieur David Lesbarrères Dean, Faculty of Graduate Studies Doyen intérimaire, Faculté des études supérieures ACCESSIBILITY CLAUSE AND PERMISSION TO USE I, Kristen Sheffield, hereby grant to Laurentian University and/or its agents the non-exclusive license to archive and make accessible my thesis, dissertation, or project report in whole or in part in all forms of media, now or for the duration of my copyright ownership. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also reserve the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that this copy is being made available in this form by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. ii

3 Thesis&Abstract& Thesurvivalofneuronsinboththecentralandperipheralnervoussystems dependonabalancebetweenpro8survivalandpro8deathsignalingpathways. Neurotrophinsandtheirprecursors,proneurotrophins,areauniquefamilyof solublesignalingproteins,whichacttopreservethisbalance.dysregulationofthese proteins,however,hasbeenimplicatedinseveralpathologies.specifically,both nervegrowthfactor(ngf)andpro8nervegrowthfactor(prongf)havebeen implicatedinthedevelopmentandprogressionofneurodegenerativediseases,such asparkinson sandalzheimer sdiseases,throughp75 NTR 8mediatedapoptotic signaling.therefore,theidentificationofsmallmoleculeinhibitorscapableof bindingtoandmodulatingngfandprongfmaybeoftherapeuticinterest.this thesisaimstocharacterizetheinhibitoryactionofpreviouslyreportedsmall molecule8basedngfinhibitors,ale80540,pd90780,ro ,andpqc083,as wellasnovelderivativesofthesecompounds,usingsurfaceplasmonresonance (SPR)spectroscopy.Oftheestablishedinhibitors,PD90780offeredthemost effectiveinhibitoryactionforbothngfandprongfbindingtop75 NTR,suggesting thatthiscompoundmayoffermultipotentinhibition.further,severalnovelligand specificmoleculeswereidentified,whichselectivelyinhibitedeitherngfor prongf8p75 NTR interactions.specifically,bvnp1,bvnp6,andbvnp7wereshown tobeeffectiveinhibitorsofngf8p75 NTR interactions,whilebvnp3andbvnp4 werefoundtobeselectivefortheinhibitionofprongfbindingtop75 NTR.The findingsofthisthesisoffernewinsightintotheuseofsmallmoleculeinhibitorsto iii

4 blockbothneurotrophinandproneurotrophin8mediatedsignaling.inaddition,the compoundsdescribedhereinmaybeusedforfutureleaddevelopment. Keywords:nervegrowthfactor,pro8nervegrowthfactor,surfaceplasmon resonance,p75 NTR,TrkA,biosensor,inhibition iv

5 Acknowledgements& Iwouldliketoexpressmygratitudeformysupervisor,Dr.GregoryRoss, whoseguidance,expertise,andpatiencehaveallowedmetodevelopthe independence,resourcefulness,andperseverancenecessaryforgraduatestudies. ThelessonsIhavelearnedunderhistutelagehavebeeninvaluable.Iwouldalsolike tothankdr.ashleyscottanddr.tomkovalafortheircontinuedinterest, encouragement,andinsight.iamforevergratefulfortheirsupport. Next,withoutthereassuranceandhelpofmylabmates,thecompletionof thisthesiswouldnothavebeenpossible.inparticular,iwouldliketothankallison KennedyforherhelpfulsuggestionsandCoreyLaamanenforhisremarkable troubleshootingabilitiesandeditorialassistance. Iwouldalsoliketothankmyfamilyfortheircontinuedsupportoverthe courseofmyacademiccareer.ioweeternalgratitudetomymother,laurie,for beingastrongrolemodelwhosequalitiesiaimtoemulate.imustalsothankmy brother,nick,forhiscomedicreliefintimesofstressandforhisunrelenting support.finally,imustacknowledgemypartnerandbestfriend,mitch,forhis endlesslove,thoughtfuladvice,andunwaveringconfidenceinmyabilities. v

6 Table&of&Contents& ThesisAbstract...iii Acknowledgements...v ListofTables...vii ListofFigures...viii ListofAbbreviations ix Chapter1:Introduction...1 Chapter2:HypothesisandObjectives...7 Chapter3:Characterizingnervegrowthfactor8p75 NTR interactionsandsmall moleculeinhibitionusingsurfaceplasmonresonancespectroscopy...9 Abstract...10 Introduction...11 MaterialsandMethods...14 ResultsandDiscussion...18 Conclusions...28 Chapter4:Usingsurfaceplasmonresonancespectroscopytocharacterizethe inhibitionofngf8p75 NTR andprongf8p75 NTR interactionsbysmallmolecule inhibitors...29 Abstract...30 Introduction...31 MaterialsandMethods...35 Results...39 Discussion...50 Conclusions...55 Chapter5:ConclusionsandFutureDirections...56 References...61) vi

7 List&of&Tables& Table3.1:IC50dataforsmallmoleculeinhibitionofNGF8p75 NTR andngf8trka interactions...27 Table4.1:IC50dataforsmallmoleculeinhibitionofNGF8p75 NTR andprongf8p75 NTR interactions...49 vii

8 List&of&Figures& Figure1.1:TheeffectofNGFonaneight8day8oldchickembryo...1 Figure3.1:AffinityofNGFforp75 NTR andtrkareceptors...19 Figure3.2:PreviouslyreportedsmallmoleculeNGFantagonistsshareconserved molecularfeatures...20 Figure3.3:NGFantagonistreceptorbindingandinhibition...22 Figure3.4:InhibitionofNGFbindingtop75 NTR andtrkabyknownngfantagonists PD90780,ALE80540,Ro ,andPQC Figure4.1:AffinityofNGFandproNGFforthep75 NTR receptor...40 Figure4.2:PreviouslyreportedsmallmoleculeNGFinhibitorsshareremarkable structuralhomology...41 Figure4.3:NovelbivalentnaphthalimidederivativesofALE Figure4.4:Smallmoleculebindingtothep75 NTR receptoranduncoupledreference flowcell...44 Figure4.5:TheinhibitionofNGF8p75 NTR andprongf8p75 NTR interactionsbyknown andnovelinhibitors...47 Figure4.6:TheinhibitionofNGFandproNGFbindingtop75 NTR bybothknownand novelinhibitors...48 & viii

9 List&of&Abbreviations& ALS Amyotrophiclateralsclerosis BDNF Brain8derivedneurotrophicfactor CI Confidenceinterval CNS Centralnervoussystem EDC 18ethyl838(38dimethylaminopropyl)carbodiimidehydrochloride EDTA Ethylenediaminetetraaceticacid EGF Epidermalgrowthfactor FDA HEPES Foodanddrugadministration (48(28hydroxyethyl)818piperazineethanesulfonicacid) IC 50 Half8maximalinhibitoryconcentration KD Affinityconstant NGF Nervegrowthfactor NHS NT83 NT84/5 p75 NTR N8hydroxysuccinimide Neurotrophin83 Neurotrophin84/5 p75neurotrophinreceptor PNS Peripheralnervoussystem ProNGF Pro8nervegrowthfactor RU Responseunit SPR Surfaceplasmonresonance TNF Tumournecrosisfactor Trk Tropomyosin8receptorkinase ix

10 1 Chapter&1:&Introduction& Nervegrowthfactor(NGF)wasfirstdescribedbyRitaLevi8Montalciniin 1951asaresultofaseriesofexperimentsexaminingtheresponseofsensoryand sympatheticgangliaofchickembryosuponexposuretotumortissue(levi8 Montalcini&Hamburger,1951).Itwasobservedthatasolublesignalingprotein (laternamedngf)releasedbythetumorpromotedthedevelopmentofneuronal projectionsfromthesensoryganglia(fig.1.1)(levi8montalcini&hamburger,1951; Levi8Montalcini,1952;Cohenetal.,1954).Forthisdiscovery,Levi8Montalciniwas awardedthenobelprizeinphysiologyormedicinein1986(bartowskaetal.,2010). Figure)1.1:)The)effect)of)NGF)on)an)eight:day:old)chick)embryo.Leftpanel:Gangliawerecultured inamediumcontainingnongf.rightpanel:gangliawereculturedinamediumcontaining10 ng/ml.thisfigurewasoriginallypublishedinscientificamerican(levi8montalcini&calissano, 1979). IncollaborationwithStanleyCohen,Levi8MontalcinidevelopedseveralNGF8 antibodieswithwhichtheywereabletodemonstratethefunctionalsignificanceof NGFinthedevelopmentofsensoryandsympatheticganglia(Bartowskaetal., 2010).FurtherworkbyCohenleadtothediscoveryofothersolublesignaling

11 2 proteins,includingepidermalgrowthfactor(egf),forwhichhewasaco8winnerof thenobelprizeinphysiologyormedicinein1986(cohen,2008). Inthefollowingdecades,severalnovel,yetstructurallyhomologousproteins, werediscoveredandtermedneurotrophins.membersoftheneurotrophicfactor familyincludengf,brain8derivedneurotrophicfactor(bdnf),neurotrophin83(nt8 3),andneurotrophin84/5(NT84/5)(Bardeetal.,1982;Philipsetal.,1990;Ibanezet al.,1993). Thefourmembersoftheneurotrophinfamilymodulateadiverserangeof functionsandsignalwithexceptionalspecificity.ingeneral,neurotrophinsregulate cellproliferation,differentiation,andsurvivalinboththecentralandperipheral nervoussystems(caietal.,2014).theuniquesignalingpropertiesofeach neurotrophinarearesultofdifferentialtropomyosin8receptorkinase(trk) activation(hempstead,2014).forinstance,ngfsignalsthroughtrka,bdnfand NT84exerttheircellulareffectsthroughTrkB,andNT83bindsspecificallytoTrkC (Chaoetal.,2006).Inaddition,eachneurotrophiniscapableofbindingthecommon neurotrophinreceptor,p75 NTR,amemberofthetumornecrosisfactor(TNF) receptorsuperfamily(chaoetal.,2006). Neurotrophinsareinitiallysynthesizedfromlargerprecursorsknownas proneurotrophins,whichconsistofann8terminalprodomainandac8terminal maturedomain(hempsteadetal.,2006).proneurotrophinshavebeenfoundtoact asdistinctligandswithbiologicalfunctionsbeyondtheirrolesasprecursors; includingtheabilitytoopposetheactionsofmatureneurotrophins(fahnestocket

12 3 al.,2004;hempstead,2014).further,theseproteinsbindareceptorcomplex consistingofp75 NTR andsortilin,amemberofthevps10p8domainreceptorfamily (Nykjaeretal.,2004). Thedysregulationofmostwidelystudiedmemberoftheneurotrophin family,ngf,inadditiontoitsprecursor,prongf,havebeenimplicatedinseveral pathologies.forinstance,ngflevelsareelevatedinseveralpainfulconditions,such asarthritis,cystitis,andchronicheadaches(aloeetal.,1992;hallidayetal.,1998; Loweetal.,1997;Oddiahetal.,1998;Sarchiellietal.,2001).Inaddition,NGF administrationhasbeenshowntoproduceallodynia,whichisdefinedaspain resultingfromastimulusthatdoesnotnormallyproducepain,inadditionto causinghypersensitivity(dycketal.,1997).further,ngfdysregulationhasbeen associatedwithneurodegenerativediseases,suchasalzheimer sdisease,asithas beenshownthatngfsignalingthroughthep75 NTR receptorinducesapoptosis leadingtoneuronaldeath(chaoetal.,2006;kenchappaetal.,2010). ProNGFalsoactsasanapoptoticligandthroughp75 NTR 8mediatedsignaling andhasbeenreportedtoinduceapoptoticsignalinginneurons,smoothmuscle cells,andoligodendricytes(beattieetal.,2002;leeetal.,2001;songetal.,2010). Interestingly,proNGFhasalsobeenshowntocauseapoptosisinbasalforebrain cholinergicneurons,suggestinginvolvementinthedevelopmentand/or progressionofalzheimer sdisease(al8shawietal.,2008).insupportofthis, elevatedlevelsofprongfhavebeenfoundinbothalzheimer sdiseasedbrainsand inanimalmodelsofalzheimer sdisease(fahnestocketal.,2001;pedrazaetal.,

13 4 2005;Perezetal.,2011).Furthermore,otherneurodegenerativediseases,suchas amyotrophiclateralsclerosis(als),parkinson sandhuntington sdiseases,have beenassociatedwithbothngfandprongfdysregulation(chenetal.,2008;allen etal.,2013;caietal.,2014).therefore,therapeuticstrategiestoinhibitbothngf andprongfp75 NTR 8mediatedsignalingmaybeofsignificantclinicalinterest. Currently,therearevariousstrategiesfortheinhibitionofNGFandproNGF. OneapproachtotheinhibitionofproNGFfocusesonmodulatingthep75 NTR receptor,asopposedtotheprecursorproteinitself.forexample,asmall, nonpeptidep75 NTR ligand,lm11a831,hasbeenreportedtopreventprongf8 induceddeathandreversethecholinergicneuritedystrophyinalzheimer sdisease mousemodelsthroughbindingthep75 NTR receptor(simmonsetal.,2014).another strategyaimedatblockingprongf8mediatedsignalinginvolvesreducingthe amountofprongfsynthesizedinthecentralnervoussystem.studieshaveshown thattheadministrationofminocycline,aderivativeoftetracycline,significantly reducesprongfproductioninmicrogliathroughitsabilitytoinhibitthe phosphorylationofp38mapk(yuneetal.,2007). Historically,drugdiscoveryeffortstoinhibitNGF8mediatedsignalinghave focusedoninhibitingitsinteractionwiththetrkareceptor.oneexampleofthisis theuseofngf8mimeticpeptides,whichcompetitivelybindtothetrkareceptor; however,theydonotelicitdownstreamngf8mediatedsignaling.whilethese peptide8basedstrategiesshowbiochemicalpromise,theyhavenotyetbeen successfulinclinicalsettings(eibletal.,2012).anotherapproachtotheinhibition

14 5 ofngfistheuseofhumanizedmonoclonalantibodies.oftheseantibody8mediated therapeutics,tanezumab,developedbypfizer,isperhapsthemostsuccessfulwith initialclinicaltrialsdemonstratingefficacyinthereductionofpainand improvementinjointfunction(laneetal.,2010).althoughtanezumabinitially demonstratedpromisingtherapeuticpotentialforthetreatmentofchronicpain, therehavebeenanumberofsafetyconcerns,includingadversechangestothe sympatheticnervoussystemandosteonecrosisofjointtissue.duetothe unfavorablesideeffectsassociatedwiththeuseofthistherapeutic,thefoodand DrugAdministration(FDA)suspendedfurtherclinicalinvestigationsofthis compound(cattaneo,2010;garber,2011).earlierthisyear,however,partial clinicalholdsonthisantibody8mediatedtherapywerelifted. Whileantibody8mediatedtherapies,suchasTanezumab,havebecomeof growingclinicalinterest,thesetherapeuticsremainbothtechnicallyand economicallychallengingtoproduce.furthermore,thesetherapiesremainhighly specificandareunabletocrosstheblood8brainbarrier.conversely,small molecules,anincreasinglypopularapproachtotheinhibitionofngf8receptor interactions,haveseveralpharmacologicaladvantages,includingthattheyare generallyeasytosynthesize,arecost8effective,andbenefitfromoralactivity. Smallmoleculeinhibitorshavebeenpreviouslydescribedandhaveshown therapeuticpotentialfortheinhibitionofngf8mediatedsignaling(jaenetal.,1995; Owolabietal.,1999;Niederhausseretal.,2000;Colquhuonetal.,2004;Heftietal., 2006;Eibletal.,2010;Eibletal.,2013).However,thereexistsagapinthescientific

15 6 researchsuchthattheseinhibitorshaveonlybeeninvestigatedfortheirabilityto inhibitngf8trkainteractions.interestingly,thecharacterizationoftheabilityof thesemoleculestoblockngf8p75 NTR interactionshasnotbeenperformed. Furthermore,withregardstotheinhibitionofproNGF,nosmallmolecule8based approacheshavebeeninvestigated. Inthisthesis,Icompletedaseriesofstudieswiththeobjectiveof characterizingtheabilityofbothknownandnovelcompoundstoinhibitngfand prongf8p75 NTR interactionsusingsurfaceplasmonresonance(spr)technology, whichisabiosensingtoolthatoffershighlysensitive,label8free,real8timeanalysis; characteristicswhichmakeitanextremelysuitabletoolforthecharacterizationof smallmolecule8basedngfinhibitors(willander&al8hilli,2009;piliariketal., 2009).Ibeginbyevaluatingtheabilityofpreviouslyreportedsmallmolecule inhibitors(ale80540,pd90780,ro ,andpqc083)toblockngfbindingto bothp75 NTR andtrkareceptorsandpresentinhibitionprofilesforeachcompound intheformofhalf8maximalinhibitoryconcentrations(ic50).next,iexplorethe abilityoftheseknowncompoundstoblockprongf8p75 NTR interactions,inaddition tocharacterizingtheinhibitionofbothngfandprongf8p75 NTR interactionsby novelbivalentnaphthalimidederivatives.

16 7 Chapter&2:&Hypothesis&and&Objectives& BothNGFandproNGFbindthecommonneurotrophinreceptorp75 NTR.The dysregulationofeachoftheseproteinshasbeenimplicatedinseveralpathological diseasestates,includingneurodegenerativediseaseslinkedtop75 NTR 8mediated apoptoticsignaling.therefore,theidentificationofmechanismstomodulateboth NGFandproNGFsignalingthroughthep75 NTR receptormaybeoftherapeutic interest. Smallmolecule8basedinhibitorswhichbindtoandmodulateNGFhavebeen describedpreviously.thesecompounds,however,haveneverbeeninvestigatedfor theirabilitytoblockeitherngforprongf8p75 NTR interactions.therefore,the centralaimofthisthesisistocharacterizeandexplorethemechanismsbywhich establishedsmallmolecule8basedngfinhibitors,aswellasnovelbivalent naphthalimidederivativesofthesecompounds,modulatebothneurotrophinand proneurotrophinsignalingusingsurfaceplasmonresonance(spr)spectroscopy. Hypothesis:Bothestablishedandnovelsmallmoleculechemicalentitiescaninhibit NGFandproNGFfrombindingtothep75 NTR receptor.

17 8 Thishypothesiswillbeevaluatedbyinvestigatingthefollowingexperimental objectives: Objectives:) 1) DeterminetheaffinityofNGFandproNGFforthep75 NTR receptor. 2) AssesstheselectivityofthesmallmoleculesforNGFbyexaminingwhether ornottheybindtothep75 NTR receptor. 3) EvaluatepercentinhibitionofbindingbetweenNGF/proNGFandp75 NTR by thesmallmolecules. 4) Determinethehalf8maximalinhibitoryconcentration(IC50)valuesofthe smallmoleculesforbothngfandprongfbindingtothep75 NTR receptor..

18 9 Chapter&3:&Characterizing&nerve&growth&factorFp75 NTR & interactions&and&small&molecule&inhibition&using&surface&plasmon& resonance&spectroscopy& Characterizing)nerve)growth)factor:p75 NTR )interactions)and)small)molecule) inhibition)using)surface)plasmon)resonance)spectroscopy) (OriginalResearch) KristenS.A.Sheffield,AllisonE.Kennedy,JohnA.Scott,GregoryM.Ross [PublishedinAnalytical*Biochemistry]

19 10 Abstract& Nervegrowthfactor(NGF)iscriticalfortheproliferation,differentiation,and survivalofneuronsthroughitsbindingtothep75 NTR andtrkareceptors. DysregulationofNGFhasbeenimplicatedinseveralpathologiesincluding neurodegeneration(i.e.parkinson sandalzheimer sdisease)andboth inflammatoryandneuropathicpainstates.therefore,smallmoleculeinhibitorsthat blockngf8receptorinteractionshavesignificanttherapeuticpotential.small moleculeantagonistsale80540,pd90780,ro ,andpqc083haveallbeen reportedtoinhibitngffrombindingthetrkareceptor.interestingly,the characterizationoftheabilityofthesemoleculestoblockngf8p75 NTR interactions hasnotbeenperformed.inaddition,theinhibitoryactionofthesemoleculeshas neverbeenevaluatedusingsurfaceplasmonresonance(spr)spectroscopy,which hasbeenproventobehighlyusefulindrugdiscoveryapplications.inthepresent study,weusesprbiosensorstocharacterizethebindingofngftothep75 NTR receptor,inadditiontocharacterizingtheinhibitorypotentialoftheknownngf antagonists.theresultsofthisstudyprovidethefirstevaluationoftheabilityof thesecompoundstoblockngfbindingtop75 NTR.Inaddition,onlyPD90780was effectiveatinhibitingtheinteractionofngfwithp75 NTR,whichsuggestsreceptor selectivitybetweenknownngfinhibitors.

20 11 Introduction& Neurotrophinsareauniquefamilyofsolublesignalingproteins,whichactto influencetheproliferation,differentiation,andsurvivalofneuronsinthecentral andperipheralnervoussystems(caietal.,2014).nervegrowthfactor(ngf),the mostwidelystudiedmemberofthisproteinfamily,interactswithtwoseparate receptorclasses;theselectivetropomyosin8receptorkinase,trka,andthecommon neurotrophinreceptor,p75 NTR (Pattarawarapan&Burgess,2003). TheinteractionofNGFwithTrkAdemonstratesaffinityinthehighpicomolar rangeandresultsinautophosphorylationofthereceptorleadingtoadownstream signalingcascadepromotingneuronalsurvival(pattarawarapan&burgess,2003; Teng&Hempstead,2004;Dray,2008).Conversely,NGF8p75 NTR interactionsare characterizedbylownanomolaraffinityandmayleadtobothpro8apoptoticand pro8survivalsignaling(pattarawarapan&burgess,2003).whilengfiscapableof bindingbothreceptors,whenbothreceptorsareexpressed,itsaffinityfortrkais influencedbythepresenceofthep75 NTR receptor.forinstance,p75 NTR hasbeen showntoenhancetheexpressionoftrka,inadditiontoincreasingitsbinding affinityforngf(massaetal.,2006;mochetti&brown,2008). NGFdysregulationhasbeenimplicatedinseveralpathologies.Forinstance,both inflammatoryandneuropathicpainstateshavebeenassociatedwithngf dysregulationandincreasedtrka8mediatedsignaling(chaoetal.,2006;dray,2008; Pezet&McMahon,2006).Likewise,increasedlevelsofNGFhavebeendetectedin thecerebrospinalfluidofpatientssufferingfromparkinson sdisease(nagatsuetal,

21 )andimprovementsinthedeliveryofNGFhavebeenshowntoameliorate cognitivefunctioninpatientswithalzheimer sdisease(tuszynskietal.,2005).it follows,then,thattherapeuticstrategiestoinhibitngf8receptorinteractionshave becomeofclinicalinterest. OnesuchapproachtoblockingNGFfrombindingtoitsreceptorsissmall moleculeinhibitors.theseinhibitorsbindtoandmodulatengf,asopposedto bindingtothetrkaandp75 NTR receptors.severaloftheseantagonistshavebeen discoveredanddescribedintheliterature(jaenetal.,1995;owolabietal.,1999; Niederhauseretal.,2000;Colquhounetal.,2004;Heftietal,2006;Eibletal.,2013). Interestingly,whilethesemoleculesareknownNGF8TrkAinhibitorsandhavebeen reportedtoinhibittrkaphosphorylationandtrka8mediateddownstreamsignaling cascades,theirabilitytoblockngffrominteractingwithp75 NTR hasnotyetbeen assessed.further,ofthepreviouslydescribedngf8bindingagents,nonehaveever beeninvestigatedusingsurfaceplasmonresonance(spr)technology,whichisa tooldesignedtoinvestigatebiomolecularinteractionsandhasbeenproventobe usefulindrugdiscoveryapplications(myszka&rich,2000;huber&mueller,2006; Minunni&Bilia,2009).SPRbiosensorsofferhighlysensitive,label8free,real8time analysis,whichmakesthemextremelysuitableforthecharacterizationofsmall molecule8basedngfantagonists(willander&al8hilli,2009;piliariketal.,2009). Inthepresentstudy,weuseSPRtechnologytocharacterizethebindingofNGFto boththetrkaandp75 NTR receptors,inadditiontocharacterizingtheinhibitory potentialofknownngfantagonistspd90780,ale80540,ro ,andpqc083.

22 13 WepresentaffinitiesofNGFtoeachofitsreceptorsdeterminedthroughsteady8 stateaffinityanalysisusingsprspectroscopy.furthermore,weassessthereceptor selectivityoftheknownngfantagonistsbyexaminingtheirabilitytoinhibitngf frombindingboththep75 NTR andtrkareceptors.inhibitionprofilesforeach inhibitorintheformofspecificityforngfandhalf8maximalinhibitory concentrations(ic50)obtainedthroughnovelmethodologyarepresented.

23 14 Materials&and&Methods& Materials* SeriesSCM5sensorchips,HEPES(48(28hydroxyethyl)818 piperazineethanesulfonicacid)bufferedsalinewithedta (ethylenediaminetetraaceticacid)andsurfactantp20(hbs8ep)buffer(0.001m HEPESpH7.4,0.15MNaCl,3mMEDTA,and0.005%v/vsurfactantP20), immobilizationbuffers(sodiumacetate,ph4.5andph5.5),aminecoupling reagents(18ethyl838(38dimethylaminopropyl)carbodiimidehydrochloride(edc),n8 hydroxysuccinimide(nhs),1.0methanolamine8hcl,ph8.5,andregeneration solutions(glycine8hcl,ph2.0)werepurchasedfromgehealthcarelifesciences (Mississauga,ON,Canada).Carrier8freeTrkAandp75 NTR wereobtainedfromr&d Systems(Minneapolis,MN,USA).MouseNGFwasobtainedfromCedarlaneLabs (Burlington,ON,Canada).PD90780,ALE80540,Ro ,andPQC083were synthesizedbysussexresearch(ottawa,on,canada).pierceelutionbufferand sodiumchloridewerepurchasedfromfisherscientific(ottawa,on,canada). SPR*and*preparation*of*sensor*surfaces* AllexperimentswerecarriedoutusingaBiacoreT200SPRspectrometer purchasedfromgehealthcarelifesciences(mississauga,on,canada).priorto immobilization,phscoutingexperimentswereconductedinordertodeterminethe optimalphfortheimmobilizationofboththep75 NTR andtrkareceptors.itwas concludedthat10mmsodiumacetateph4.5and5.5yieldedthegreatestpre8 concentrationeffectforp75 NTR andtrkaimmobilizations,respectively.

24 15 Immobilizationexperimentsinvestigatingtheoptimalbindinglevelsforboth receptorswereconducted,allowingfortheselectionofanimmobilizationlevel between ru;increasedproteinloadinginterferedwithbindingkinetics,as dissociationconstantwereinconsistent.thecm5sensorchipwasactivatedby injectingamixtureof0.2medcand0.05mnhsataflowrateof5µl/minfor7 minutes.p75 NTR wasdilutedto10µg/mlin10mmsodiumacetatebufferph4.5 andimmobilizeduntilalevelbetween850and950relativeresponseunits(ru) (~15fmol/mm 2 )wasreached.excessreactiveestersonthesensorchipsurface weredeactivatedwith1methanolamineph8.5ataflowrateof5µl/minfor7 minutes.thetrkareceptorwasimmobilizedfollowingtheaboveprocedure; however,10mmsodiumacetateph5.5immobilizationbufferwasused.inthe TrkAimmobilization,alevelbetween850and950RU(~11fmol/mm 2 )was reached.flowcellsusedforreferencewereactivatedandblockedasdescribedfor thep75 NTR andtrkaimmobilizations;however,remaineduncoupled.bindingwas expressedasrelativeresponseunits(ru),whichisdefinedastheresponseobtained fromtheflowcellscontainingtheimmobilizedreceptorsminustheresponse obtainedfromthereferenceflowcells. Affinity*assays*for*NGF*binding*to*p75 NTR* and*trka*receptors* AffinityofNGFtop75 NTR andtrkawasdeterminedusingaserialdilution series.ngfwasdilutedinhbs8epbufferwithconcentrationsrangingfrom nmto50nmandalloweda60secondcontacttimefollowedbya120second dissociationphase.forexperimentsinvolvingp75 NTR,thesensorchipsurfacewas

25 16 regeneratedwitha15secondinjectionofasaltcocktail(2:1v/vpierceelution bufferand4mnacl)previouslydescribedbyabdicheetal.(2008).inexperiments withtrka,a15secondinjectionofglycine8hclph2.0wasusedforsensorsurface regeneration. *Interactions*of*small*molecules*with*p75 NTR* and*trka*receptors** SmallmoleculesweredilutedinHBS8EPbufferataconcentrationof50µM andinjectedovertheimmobilizedp75 NTR andtrkareceptorsfor60seconds.))all compoundswereallotteda120seconddissociationtime.receptorbindingwas determinedbyassessingthebindingresponseofeachcompoundinrus(1ru= 1pg/mm 2 ).Thesensorchipsurfaceswereregeneratedasdescribedabove.) *Inhibition*of*NGF*binding*p75 NTR* and*trka*receptors*by*small*molecules SmallmoleculesweredilutedinHBS8EPbufferataconcentrationof50µM andpre8incubatedfor1hourwith10nmngfbeforeinjectionovertheimmobilized receptors.controlsamples(withnoaddedinhibitor)wereusedandyieldedthe maximalbindingresponse.percentinhibitionoftheprotein8receptorinteraction wasdeterminedbyassessingthecompoundbindingresponseinrelationtothe controlsampleresponse.thesensorchipsurfaceswereregeneratedasdescribed above. IC50*determination* SmallmoleculesweredilutedinHBS8EPbufferatvaryingconcentrations rangingfrom320µmto3.2nm,correspondingtoequalspacingonalogarithmic

26 17 scale.compoundswerepre8incubatedfor1hourwith10nmofngfbefore injectionovereithertheimmobilizedp75 NTR ortrka.dose8responsecurveswere generatedandwereusedforthedeterminationofthehalf8maximalinhibitory concentrations(ic50)forallcompounds. Data*analysis* TransformationofdataforNGF8receptoraffinityanalyses,smallmolecule receptor8interactions,andinhibitionexperimentswereperformedwithbia evaluationsoftwarefromgehealthcarelifesciences(mississauga,on,canada). Thegenerationofdose8responsecurvesanddeterminationofIC50valueswere conductedusingprismgraphpad6.0(lajolla,ca,usa).

27 18 Results&and&Discussion& Determination*of*the*affinity*of*NGF*to*p75 NTR *and*trka*receptors* TheaffinityofNGFforbothp75 NTR andtrkareceptorswasdetermined throughsteady8stateanalysis.aconcentrationseriesrangingfrom0.0125nmto50 nmwasrunovereachimmobilizedreceptor(figs.3.1a,3.1b).theconcentrations chosenforthisdeterminationwerebasedonresultsfromscreeningexperimentsin whichngfsaturatedtheimmobilizedreceptorsonthesensorsurface.usingthe BIAevaluationsoftware,theresponseofNGFwasplottedagainstconcentrationfor eachconcentrationofngfusedinthedilutionseries(figs.3.1c,3.1d).datafor bothp75 NTR andtrkawasevaluatedusinga1:1bindingmodel.bindingofngfto p75 NTR yieldedahigherresponsethanthatobservedwithtrka(figs.3.1a,3.1b). Thismaybeduetothefactthatthroughtheaminecouplingproceduredescribed previously,trkawasimmobilizedinsuchawaythatpreventedthesameamountof NGFbinding.Inaddition,theaffinity(KD)ofNGFforp75 NTR (12.9±0.5nM)was determinedtobesimilartotheaffinityofngffortrka(15.0±3.7nm).previously, theaffinitiesofngfforitsreceptorshavebeendeterminedusingkineticstudies andsprspectroscopy,andwerereportedtobe1nmforngf8p75 NTR interactions and2nmforngf8trkainteractions(nykjaeretal.,2004).whiletheaffinities obtainedthroughkineticstudiesarelowerthanthoseobtainedthroughoursteady8 stateanalyses,ourreportsareconsistentwiththepreviouslyreportedaffinity constantsinthattheaffinityofngfforp75 NTR andtrkainteractionswereina similarrange.thedifferencesinthemagnitudeofvaluesmaybeduetothediffering

28 19 methodologyusedfortheirdetermination.further,ourdatasupportsthenotion thattheaffinitiesofngftop75 NTR andtrkaaresimilarunlessp75 NTR isco8 expressedwithtrkatoelicithigheraffinitybindingofngfandtrka(hempsteadet al,1991;rossetal.,1998;tonietal.,2014). ) A 200 B 60 Response (RU) Response (RU) C Time (s) 150 K D = 12.9 ± 0.5 nm D 30 Time (s) K D = 15.0 ± 3.7 nm Response (RU) Concentration, nm Concentration, nm ) Figure)3.1:)Affinity)of)NGF)for)p75 NTR )and)trka)receptors.)concentrationseriesofngf (0.0125nMto50nM)wererunoverp75 NTR (A)andTrkA(B)andusedtocalculatetheaffinity ofngfforeachreceptorthroughsteady8stateanalysis.responseofngfbindingtoboth p75 NTR (C)andTrkA(D)wasplottedversusconcentrationforeachconcentrationusedinthe dilutionseries.theaffinity(k D)ofNGFforp75 NTR andtrkawasdeterminedtobe12.9±0.5 nmand15.0±3.7nm,respectively.datapointsarepresentedasmeanvaluesoftriplicate measures.errorbarsarerepresentedasstandarderrorofthemeanwithann8value=3. ) * *Interactions*of*small*molecules*with*p75 NTR* and*trka*receptors Response (RU) ) Previouslydescribedsmallmolecule8basedNGFantagonists,ALE80540, PD90780,Ro ,andPQC083,havebeensuggestedtoshareconserved molecularfeatures(eibletal.,2013).forinstance,theseinhibitorsshareaplanar

29 20 conjugatedringsystem,incorporatingcommonamine,methyl,carbonyl,and carboxylicacidfunctionalgroups(eibletal.,2013).theloopi/ivcleftofngfisthe proposedbindingsiteforthesesmallmoleculesinhibitors,withhighimportance placedonthelysine32,phenylalanine101,andarginine100residues(eibletal., 2013).ThestructuresofthesecompoundsarepresentedinFigure3.2. A B C D Figure)3.2:)Previously)reported)small)molecule)NGF)antagonists)share)conserved)molecular) features.thestructuresofpreviouslyreportedngf8bindingantagonists(a)ale80540(b)pd90780 (C)Ro and(D)PQC083. TodeterminethespecificityofthesmallmoleculesforNGF,weassessed whethertheyalsoboundtothep75 NTR andtrkareceptors(figs.3.3a,3.3b).after injectionof50µmofeachcompoundoverboththep75 NTR andtrkasensor surfaces,itwasfoundthatale80540(0.2ru),pd90780(0.3ru),andro (0.1RU)boundneitherthep75 NTR northetrkareceptorincomparisontoablank injectionofbufferwithnoaddedsmallmolecule(p>0.05).pqc083,ontheother

30 21 hand,wasfoundtobindsignificantlytobothp75 NTR) (18RU;P=0.0006)andTrkA (42RU;P=0.0003).WeattributethepositivebindingofPQC083tomultiple proteinstothehydrophobicnatureofthecompound,makingitunsuitableforusein SPRassays.ThesefindingssuggestthatALE80540,PD90780,andRo exhibitspecificbindingtongfasopposedtothep75 NTR andtrkareceptors, whereaspqc083displaysnon8specificbinding.

31 22 A Response (RU) [Inhibitor]= 50 µm B * 50 * Response (RU) [Inhibitor]= 50 µm 0 ALE-0540 PD90780 Percent Inhibition (%) Ro Inhibitor PQC 083 C * * * 0 ALE-0540 PD90780 Ro Inhibitor p75 NTR TrkA PQC ALE-0540 PD90780 Ro PQC 083 Inhibitor Figure)3.3:)NGF)antagonist)receptor)binding)and)inhibition.TheknownNGFantagonistswere runoverp75 NTR (A)andTrkA(B)todetermineifbindingisspecifictoNGF.Theantagonistswere thenincubatedwith10nmofngfandrunoverp75 NTR (C)andTrkA(D)toassesstheinhibitory potentialofeach.dataarepresentedasmeanvaluesoftriplicatemeasureswithstandarderrorof themeanbarswithann8value=3.significantdifferenceisdenotedbyanasterisk. *

32 23 Inhibition*and*determination*of*halfHmaximal*inhibitory*(IC50)*concentrations* Anevaluationoftheinhibitorypotentialofeachsmallmoleculewas conductedbyincubatingeachcompoundat50µmwith10nmofngfforonehour (Figs.3.3C,3.3D).ThebindingresponseswerethenassessedusingSPRand comparedtocontrolsamplesof10nmofngfwithnoaddedinhibitortogenerate percentinhibitionofmaximalbindingvaluesforeachsmallmolecule.following this,wegeneratedaseriesofdose8responsecurvesinordertodeterminethehalf8 maximalinhibitoryconcentrations(ic50)ofeachcompound(fig.3.4).theic50 valuesarepresentedintable3.1foreaseofuseandinterpretation.combined, thesestudiesofferanovelapproachtothecharacterizationofsmallmolecule antagonists,inadditiontoallowingforthedeterminationofthespecificityand selectivityoftheknownngfinhibitorsforbothngf8p75 NTR andngf8trka interactions.

33 24 A B Percent Response (%) Percent Response (%) [NGF]= 10 nm log [Inhibitor], µm [NGF]= 10 nm log [Inhibitor], µm ALE-0540 PD90780 Ro ALE-0540 PD90780 Ro Figure)3.4:)Inhibition)of)NGF)binding)to)p75 NTR )and)trka)by)known)ngf)antagonists)pd90780,) ALE:0540,)Ro)08:2750,)and)PQC)083.)(A)DetailedconcentrationresponsedemonstratesthatALE8 0540(IC 50=>300µM;R 2 <0.5),PD90780(IC 50=110µM;CI=938129µM;R 2 =0.95),andRo (IC 50=244µM;CI= µM;R 2 =0.87)inhibitsNGF8p75 NTR interactionsinthelow8to8high micromolarrange.(b)detailedconcentrationresponsedemonstratesthatale80540(ic 50=149µM; CI= µM;R 2 =0.88),PD90780(IC 50=47µM;CI=35864µM;R 2 =0.94),andRo (IC 50= 33µM;CI=19857µM;R 2 =0.87)inhibitsNGF8TrkAinteractionsinthemid8to8highmicromolarrange. Datapointsarepresentedasmeanvaluesoftriplicatemeasures.Errorbarsarerepresentedas standarderrorofthemeanwithann8value=3.ic 50valuesarepresentedwith95%confidence intervals(ci).r 2 isdefinedasthecoefficientofdeterminationandusedtoassessgoodnessoffit.

34 25 OurstudiesindicatethatALE80540hasamoderateinhibitoryeffectonNGF8 TrkAinteractions(44%)andthatitisnotastronginhibitorofNGFbindingto p75 NTR (6.2%);thisdifferentialreceptorinhibitionisstatisticallydifferent (P=0.0002).WhereasaccurateinhibitioncurvescouldnotbeplottedforNGF8 p75 NTR interactions(ic50>300µm),dose8responseassaysdemonstratethatale8 0540hasinhibitoryactioninthehighmicromolarrangeforTrkA(149µM;CI= µM).ThepreviouslyreportedvalueforNGF8TrkAinteractionsis3.7µM (Owolabietal.,1999);thediscrepancybetweenvaluesislikelyduetothe differencesintheassaysusedfordetermination.forinstance,experiments conductedbyowolabietal.(1999)utilizedpc12wholecellstudies,whereasthe assaypresentedhereeliminatescertainpossibilities,suchasreceptorinteractions. ThereisnoreportedIC50forNGF8p75 NTR interactions;however,anic50of5.8µm hasbeenreportedforngf8trka8p75 NTR (Owolabietal.,1999).Thelowmicromolar inhibitionreportedheremightbeduetothepresenceofthetrkareceptorand,as such,isnotcomparabletothengf8p75 NTR datareportedinthisstudy. PD90780wasfoundtobeaneffectiveinhibitorofNGFbindingtoTrkA (100%)andamoderateinhibitorinblockingNGF8p75 NTR interactions(63%).the inhibitoryactionofpd90780onngf8trkainteractionswasshowntobe significantlygreaterthanfortheinteractionofngfandp75 NTR (P=0.0001). Inhibitorybindingassaysrevealedmid8micromolarinhibitionofNGF8TrkA(47µM; CI=35864µM)andhighmicromolarinhibitionofNGF8p75 NTR (110µM;CI= µm).colquhuonetal.(2004)havepreviouslydescribedtheinhibitoryeffectsof

35 26 PD90780onNGF8TrkAandNGF8p75 NTR interactions,andreport1.8µmand23.1 µm,respectively.ourdataisconsistentwithpd90780havingagreaterinhibitory effectonngfbindingtrkaincomparisontop75 NTR.Incomparisontothelow micromolarvaluesreportedpreviously,ourdatafallinthemid8to8highmicromolar range,whichcouldbeduetothedifferencesinexperimentalproceduresandassays used. OurstudiesdemonstratethatRo blocksNGFfrominteractingwith TrkA(99%)significantlymorethanp75 NTR (37%)(P=0.0001).Ourdose8response studiesindicatethatro hasinhibitoryactioninthelow8to8midmicromolar range(33µm;ci=19857µm)forngf8trkaandhighmicromolarrange(244µm; CI= µM)forNGF8p75 NTR.AlthoughRo hasbeendescribed previously(niederhauseretal.,2000),therearenoreportedic50valuesforthe inhibitionofngf8receptorinteractionsintheliterature. Interestingly,PQC083demonstratedmoderateinhibitoryeffectsofNGF bindingp75 NTR (70%);however,mixedinhibitionofNGF8TrkAinteractionswas observed.forinstance,pqc083demonstratedhighnon8specificbinding(bound thereceptorintheabsenceofinhibitingngfreceptorbinding).further,the inhibitorybindingassaysprovedtobedifficulttoperformwithpqc083duetothe incompatibilityofthecompoundwithsprassays.wewere,therefore,unableto analyzedose8responsedatawhenevaluatingtheinhibitoryactionofpqc083on bothngf8p75 NTR andngf8trkainteractions;noic50valuescouldbedeterminedfor thisinhibitor.however,inpreviousstudiesconductedbyeibletal.(2012),low

36 27 micromolar(7.0µm)inhibitionofngfbindingtotrkabypqc083hasbeen reported.theinhibitorycapacityofpqc083toblockngffrominteractingwith p75 NTR,though,hasnotbeenassessed. Table)3.1:)IC 50)data)for)small)molecule)inhibition)of)NGF:p75 NTR )and)ngf:trka)interactions.) Dataarepresentedasmeanvaluesoftriplicatemeasureswithn=3.) p75 NTR TrkA ALE80540 >300µM 149µM(CI= µM) PD µM(CI=938129µM) 47µM(CI=35864µM) Ro µM(CI= µM) 33µM(CI=19857µM) PQC083 NotDetermined NotDetermined

37 28 Conclusions& ThroughtheutilizationofSPRtechnology,theeffectivenessofsmall molecule8basedngfinhibitorsale80540,pd90780,ro ,andpqc083was assessedwithregardstotheirabilitytoinhibitbindingtoboththep75 NTR andtrka receptors.pd90780wasfoundtoofferthemostpotentialfortheinhibitionofboth NGF8TrkAandNGF8p75 NTR interactions.ale80540offeredinhibitoryactionforonly NGFbindingtoTrkA,whileRo wasshowntobeaneffectiveinhibitorof NGF8TrkAbindingandonlymildlyblockedNGFfrominteractingwithp75 NTR. Conversely,PQC083yieldedexperimentalartifacts(demonstratednon8specific binding)makingitdifficulttocharacterizewithourassays.usingsprbiosensorsto characterizengf8bindingagentsoffershighlysensitive,label8free,real8time analysis.thistechnologyhasbeenproventobehighlyeffectiveindrugdiscovery applicationsandmaybeusefulforfurtheridentificationofnoveltherapeuticsthat inhibitngf8p75 NTR interactions,whichmayhaveimplicationsinthetreatmentof neurodegenerativediseases,suchasparkinson sandalzheimer sdiseases. ) )

38 29 Chapter&4:&Using&surface&plasmon&resonance&spectroscopy&to& characterize&the&inhibition&of&ngffp75 NTR& and&prongffp75 NTR & interactions&by&small&molecule&inhibitors& Using)surface)plasmon)resonance)spectroscopy)to)characterize)the)inhibition) of)ngf:p75 NTR) and)prongf:p75 NTR )interactions)by)small)molecule)inhibitors) (OriginalResearch) KristenS.A.Sheffield,RahulVohra,JohnA.Scott,GregoryM.Ross [PublishedinPharmacological*Research]

39 30 Abstract& Nervegrowthfactor(NGF),amemberoftheneurotrophinfamily,actsto influencethesurvivalanddifferentiationofneuronsinboththecentraland peripheralnervoussystemsviaitsbindingtothep75 NTR andtrkareceptors.its precursor,prongf,hasbeenshowntobethedominantformofngfinthecentral nervoussystem,suggestingabiologicalfunctionbeyonditsroleasaprecursor.like NGF,proNGFisknowntobindthep75 NTR receptor.thedysregulationofbothngf andprongfhavebeenimplicatedinseveralpathologies,including neurodegenerativediseaseslinkedtop75 NTR 8mediatedapoptoticsignaling. Therefore,theidentificationofsmallmoleculeinhibitorscapableofinhibitingboth NGFandproNGF8p75 NTR interactionsmaybeoftherapeuticinterest.inthepresent study,weexaminetheinhibitoryactionofknownsmallmolecule8basedinhibitors PD90780,ALE80540,Ro ,andPQC083,aswellasnovelderivativesofthese compounds,usingsurfaceplasmonresonance(spr)spectroscopy.

40 31 Introduction& Neurotrophinsareauniquefamilyofsolublesignalingproteins,including nervegrowthfactor(ngf),brain8derivedneurotrophicfactor(bdnf),neurotrophin 3(NT83),andneurotrophin4/5(NT84/5).Theseproteinsacttoinfluencethe survivalanddifferentiationofneuronsinthecentralandperipheralnervous systems,inadditiontobeingcriticaltobothdevelopingandmaturenervous systems(chaoetal.,2006).whilethefourmembersoftheneurotrophinfamily shareremarkablestructuralhomology,theseproteinsmodulateadiverserangeof functionsandsignalwithexceptionalspecificity(pattarawarapan&burgess,2003). Theuniquesignalingpropertiesofeachneurotrophinarearesultofdifferential tropomyosin8receptorkinase(trk)activation(huang&reichardt,2003).for instance,themostwidelystudiedmemberoftheneurotrophinfamily,ngf,signals throughtheselectivetropomyosin8receptorkinase,trka,inadditiontothecommon neurotrophinreceptor,p75 NTR (Kaplan&Miller,2000). NGF8TrkAinteractionsdemonstratepicomolaraffinity,whichresultsinthe autophosphorylationofthereceptorleadingtoadownstreamcascadeoftrk8 mediatedsignalingandthepromotionofneuronalsurvival(pattarawarapan& Burgess,2003;Teng&Hempstead,2004;Dray,2008).Conversely,NGFsignaling throughthep75 NTR receptorresultsinalownanomolarinteractionandis recognizedtobecomplex.forexample,ngf8mediatedp75 NTR signalinghasbeen implicatedinbothneuronalsurvivalandneuronaldeath,inadditiontoaxonal pruningandapoptosis(colquhounetal.,2004;he&garcia,2004).whilengfbinds toboththetrkaandp75 NTR receptors,thehighaffinitynatureofngf8trkabinding

41 32 ismediatedbythepresenceofp75 NTR (Massaetal.,2006;Mochetti&Brown,2008). Themechanismbywhichp75 NTR mediatesthengf8trkahigh8affinitystateisstill notfullyunderstood.currently,therearetwotheories;thefirstsuggeststhat p75 NTR,TrkA,andNGFformaternarycomplexresultinginahigheraffinityin comparisontowheneitherreceptorisexpressedalone,whiletheothertheory proposesthatp75 NTR actstoclusterthengftogether,facilitatingbindingtotrka (Barker,2007). Allneurotrophinsaresynthesizedfromlargerprecursorsknownas proneurotrophins,whichconsistofann8terminalprodomainandac8terminal maturedomain(wehrmanetal.,2007).theprecursorofngf,prongf,while initiallythoughttobebiologicallyinactive,hasbeenshowntobethedominantform ofngfinthecentralnervoussystem,suggestingabiologicalfunctionbeyondits roleasaprecursor(fahnestocketal.,2004).prongf,likengf,bindsthep75 NTR receptor;however,prongfalsobindssortilin,amemberofthevps10p8domain receptorfamily(nykjaeretal.,2004). TheinteractionofproNGFwithp75 NTR hasbeenshowntodemonstratelow nanomolaraffinity,whereasitsinteractionwithsortilinisofmuchhigheraffinity (Nykjaeretal.,2004).ProNGFhasbeenshowntoinduceapoptosisthroughthe activationofthep75 NTR receptor,whichfurthersupportsthenotionthatthe precursorisadistinctandbiologicallyactiveligandcapableofopposingtheactions ofthematureneurotrophin,ngf(leeetal.,2001;songetal.,2010).

42 33 DysregulationofbothNGFandproNGFhavebeenimplicatedinseveral pathologies.forexample,ngflevelsareelevatedinseveralpainfulconditions,such asarthritis,cystitis,andchronicheadaches(aloeetal.,1992;loweetal.,1997; Hallidayetal.,1998;Oddiahetal.,1998;Sarchiellietal.,2001).Inaddition,a subcutaneousinjectionofngfintotheforearmofhealthyadultswasfoundto produceallodynia,whichisdefinedaspainresultingfromastimulusthatdoesnot normallyproducepain,inadditiontocausinghypersensitivity(dycketal.,1997). Further,NGFdysregulationhasbeenimplicatedinneurodegenerativediseasestates asngf8p75 NTR mediatedsignalinginducesapoptosisleadingtoneuronaldeath (Kenchappaetal.,2010).Likewise,proNGFhasbeenassociatedwith neurodegeneration.forinstance,prongf8p75 NTR interactionsinduceapoptotic signalinginneurons,smoothmusclecells,andoligodendricytesatsubnanomolar concentrations(leeetal.,2001;beattieetal.,2002;songetal.,2010).inaddition, increasedlevelsofprongfhavebeenfoundinalzheimer sdiseasedbrains (Fahnestocketal.,2001).Therefore,therapeuticstrategiestoinhibitbothNGFand prongfsignalingmaybeofsignificantclinicalinterest. WhiletheinhibitionofNGFhasbeenwidelystudied,investigationsregarding theinhibitionofprongfarelimited.onetherapeuticapproachgainingincreased attentionistheuseofsmallmoleculeinhibitorstoblockngffrombindingthetrka andp75 NTR receptors.thesecompoundsbindngf(asopposedtothereceptors)in ordertomodulatesignalingandhavebeendescribedpreviously(jaenetal.,1995; Owolabietal.,1999;Niederhauseretal.,2000;Colquhounetal.,2004;Heftietal., 2006;Eibletal.,2010;Eibletal.,2013;Sheffieldetal.,2015).Conversely,proNGF

43 34 inhibitoryinvestigationshavefocusedonmodulatingthep75 NTR receptor(as opposedtoprongfitself),aswellasreducingtheproductionofprongfinthe centralnervoussystem(massaetal.,2006;yuneetal.,2007;al8gayyaretal.,2011). Interestingly,therapeuticsthatbindtoandmodulateproNGFhavenotyetbeen investigated. Inthepresentstudy,weusesurfaceplasmonresonance(SPR)spectroscopy tocharacterizeprongf8p75 NTR interactions.inaddition,weexaminetheinhibitory potentialofknownsmallmolecule8basedinhibitorsale80540,pd90780,ro ,andPQC083,aswellasnovelderivativesofthesecompounds.Weassessthe ligandselectivityofthesmallmoleculesthroughtheexaminationoftheirabilityto blockbothngfandprongffrombindingthep75 NTR receptor.

44 35 Materials&and&Methods& Materials SeriesSCM5sensorchips,HEPES(48(28hydroxyethyl)818 piperazineethanesulfonicacid)bufferedsalinewithedta (ethylenediaminetetraaceticacid)andsurfactantp20(hbs8ep)buffer(0.001m HEPESpH7.4,0.15MNaCl,3mMEDTA,and0.005%v/vsurfactantP20), immobilizationbuffer(sodiumacetate,ph4.5),aminecouplingreagents(18ethyl838 (38dimethylaminopropyl)carbodiimidehydrochloride(EDC),N8hydroxysuccinimide (NHS),1.0Methanolamine8HCl,pH8.5,andregenerationsolutions(glycine8HCl,pH 2.0)werepurchasedfromGEHealthcareLifeSciences(Mississauga,ON,Canada). Carrier8freep75 NTR wasobtainedfromr&dsystems(minneapolis,mn,usa). MouseNGFandrecombinanthumanproNGFwereobtainedfromCedarlaneLabs (Burlington,ON,Canada).AllsmallmoleculeinhibitorsweresynthesizedbySussex Research(Ottawa,ON,Canada). SPR*and*sensor*surface*preparation* AllexperimentswerecarriedoutusingaBiacoreT200SPRspectrometer fromgehealthcarelifesciences(mississauga,on,canada).priorto immobilization,phscoutingexperimentswereconductedinordertodeterminethe optimalphfortheimmobilizationofthep75 NTR receptoranditwasconcludedthat 10mMsodiumacetatepH4.5yieldedthegreatestpre8concentrationeffect. Immobilizationexperimentsinvestigatingtheoptimalbindinglevelsforboth receptorswereconducted,allowingfortheselectionofanimmobilizationlevel

45 36 between ru,asincreasedproteinloadinginterferedwithbindingkinetics. TheCM5sensorchipwasactivatedbyinjectingamixtureof0.2MEDCand0.05M NHSataflowrateof5µl/minfor7minutes.p75 NTR wasdilutedto10µg/mlin10 mmsodiumacetatebuffer,ph4.5andimmobilizeduntilalevelbetween850and 950relativeresponseunits(RU)(~15fmol/mm 2 )wasreached.excessreactive estersonthesensorchipsurfaceweredeactivatedwith1methanolamine,ph8.5at aflowrateof5µl/minfor7minutes.flowcellsusedforreferencewereactivated andblockedasdescribedabove,butremaineduncoupled.bindingwasexpressed asrelativeresponseunits(ru),whichisdefinedastheresponseobtainedfromthe flowcellcontainingtheimmobilizedreceptorminustheresponseobtainedfromthe referenceflowcell. *Affinity*assays*for*NGF*and*proNGFH*p75 NTR *interactions* TheaffinityofNGFandproNGFtop75 NTR wasdeterminedusingaserial dilutionseries.ngfwasdilutedinhbs8epbufferwithconcentrationsrangingfrom nMto50nMandalloweda60secondcontacttimefollowedbya120second dissociationphase.similarly,prongfwasdilutedinhbs8epbufferwith concentrationsrangingfrom0.025nmto100nmandalloweda120secondcontact timefollowedbya900seconddissociationphase.thesensorchipsurfacewas regeneratedwitha15secondinjectionofasaltcocktail(2:1v/vpierceelution bufferand4mnacl)anda15secondinjectionofglycine8hcl,ph2.0forngfand prongfexperiments,respectively.theresponseofngfandprongfobtainedfrom

46 37 eachconcentrationinthedilutionserieswasplottedagainstconcentrationvalue usingthebiaevaluationsoftwareandwasevaluatedusinga1:1bindingmodel. Interactions*of*small*molecules*with*the*p75 NTR* and*receptor* SmallmoleculesweredilutedinHBS8EPbuffertoaconcentrationof100µM andinjectedovertheimmobilizedp75 NTR receptorfor60seconds.)allcompounds wereallotteda120seconddissociationtime.receptorbindingwasdeterminedby assessingthebindingresponseofeachcompoundinrus(1ru=1pg/mm 2 ).The sensorchipsurfacewasregeneratedasdescribedabove. Inhibition*of*NGF*and*proNGF*binding*the*p75 NTR *receptor*by*small*molecules SmallmoleculesweredilutedinHBS8EPbufferatasingleconcentration(100 µm)andpre8incubatedfor1hourwitheither10nmngfor10nmprongfbefore injectionovertheimmobilizedreceptor.controlsamples(withnoaddedinhibitor) wereusedtodeterminethemaximalbindingresponse.percentinhibitionofboth NGF8p75 NTR andprongf8p75 NTR interactionsweredeterminedbyassessingthe compoundbindingresponseinrelationtothecontrolsampleresponse.thesensor chipsurfaceswereregeneratedasdescribedabove. Theaccuracyofourassaysystemistypically3%standarddeviation,and antagonistsofinterestdemonstrateligandselectivitythrougha30%orgreater differencebetweentheinhibitionofngfandprongf8p75 NTR interactions.power calculationssuggestedtriplicatemeasurementwouldallowforthedetectionof greaterthan20%inhibitionwithconfidenceofgreaterthan95%.

47 38 *IC50*determination* Half8maximalinhibitoryconcentrations(IC50)weredeterminedforsmall moleculesexhibiting50%orgreaterpercentinhibitionofmaximalbinding.small moleculesweredilutedinhbs8epbufferatvaryingconcentrationsrangingfrom 320µMto3.2nMand100µMto10nM(correspondingtoequalspacingona logarithmicscale)forngfandprongfexperiments,respectively.compoundswere pre8incubatedfor1hourwitheither10nmngfor10nmprongfbeforeinjection overtheimmobilizedp75 NTR receptor.dose8responsecurvesweregeneratedand wereusedforthedeterminationofthehalf8maximalinhibitoryconcentrations (IC50). Data*analysis* TransformationofdataforNGFandproNGF8p75 NTR affinityanalysis,small moleculereceptor8interactions,andinhibitionexperimentswereperformedwith BIAevaluationsoftwarefromGEHealthcareLifeSciences(Mississauga,ON, Canada).Thegenerationofdose8responsecurvesanddeterminationofIC50values wereconductedusingprismgraphpad6.0(lajolla,ca,usa).