Identification of selected small molecule targets in living cells using Capture Compoundsâ„¢...

Identification of selected small molecule targets in living cells using

Capture Compounds™ through a bio-orthogonal chemical ligation method

Inaugural Dissertation

to obtain the Academic Degree

Doctor rerum naturalium (Dr. rer. nat.)

Submitted to the Department of Biology, Chemistry and Pharmacy

of the Freie Universität Berlin

by

João André Banha Oliveira

from Lisbon, Portugal

November, 2015

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The following experimental work was developed between December 2010 and August 2014 atcaprotecbioanalyticsGmbHunderthesupervisionofProf.Dr.HubertKöster

1stReviewer:Prof.Dr.HubertKöster2ndReviewer:Prof.Dr.MarkusWahlDateofdefense:11April2016

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AcknowledgmentsIwouldliketoexpressmyappreciationtoProf.HubertKöster,FounderandManagingDirector

of caprotec bioanalytics GmbH, for recruiting me and giving me this challenging but excitingopportunity towork in a start-upenvironment. Thank you for your support andpositive feedbackduringourprojectdiscussions.

I would also like to acknowledge Prof. Markus Wahl at the Freie Universität Berlin for hisacceptancetoco-supervisemyresearchprojectandaidmeinnavigatingthebureaucracy.

AspecialthankyouisdedicatedtoDr.MathiasDreger,HeadoftheBiochemistryDepartmentatcaprotecthatguidedmethroughoutthisbumpyroad.Ideeplyappreciateyoualwayshavingyourdooropenfordiscussionandgivingyourbesttomotivatemetomoveforward.Ialsoneedtothankyouforyourcommitmentinreviewingthisdissertationandsharingyourthoughts.

Toallmycolleaguesat caprotecbioanalytics, some thatwere there from thebeginningandothers thatcameandwent, thankyou for thesupportaroundthe labandmeetingsbutespeciallythankyouforthefunnymoments.Laughteralwaysmakesthejobeasier.Youmademefeel lessofan outsider addressing me in English but still teaching me some German. I will never forgetLuftfeuchtigkeitandStreichholzschächtelchen.

ThereareafewcolleaguesIneedtosingleoutinmyacknowledgements.LisavonKleistfortheveryhelpfulbrainstormingsessionsbutmostofallforyourpersonalityandworkethics.YanLuoforgettingme started in the lab and later collaborating in thephenobarbital project. The guys at theMedicinal Chemistry Lab, SimonMichaelis, Frank Polster and Daniel Ohlendorf, as well as HenrikDieks for synthesizingall compounds Iused in thiswork.MatthiasHakelbergandChristianDalhofffromtheAnalyticsDepartmentforthecontinuoussupportinconfirmingthestabilityandqualityofmycompounds.KathrinBarthofortakinggoodcareofmymassspectrometrysamplesandanalyzingthemasquicklyaspossibleaswellasthefriendlydiscussionsaroundExceltablesonhowtoextractthe information from theMSdata. Finally,Michael Sefkow for the supportandenthusiasm in thiswholeproject.Thankyouall.

This work was supported by the Initial Training Network, Bio-Orthogonal Chemo-SpecificLigation (BioChemLig), funded by the FP7Marie Curie Actions of the European Commission (FP7-PEOPLE-2008-ITN-238434). Within this consortium I had the opportunity to establish greatconnectionstomyfellowcolleagues,whoIwouldliketosaluteforthefriendshipcreatedduringthemeetingsandputtingupwiththeonebiologistinthemidstofallchemists.ToMarcoandValentina,who chose to come toBerlin for their secondments, thank you for lettingme teach youaroundabiochemistrylab.Itrulyenjoyedit.Also,totheP.I.softhegroups,thankyouforyourcommentsandguidanceintheprogressreportsmeetings.

A warm appreciation goes out tomy family who have supportedme inmy journey acrossbordersandawayfromhome.Alwaysconcernedbutalsomotivatingmetokeepongoingandaimforhappiness.

Finally,IwouldnothavecomethisfarwithouttheloveandsupportofmypartnerChris.Thesepast twoyearshavebeenachallengebutablessingbecauseofyou.Weovercomeour limitationsdrawingstrengthfromeachotherandthatmakesusundefeatable.

ThankyouformakingHomewhereverandwheneverweare.

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PrefaceTheoverallaimofthisstudywastheintroductionofanovelapproachfortheidentificationof

small-moleculetargetsfromlivingcellsusingcapturecompoundmassspectrometry.Toachievethisgoal, the research was performed in a step-wise comprehensive manner that is reflected in thestructureofthisdissertation.

A general introduction covers the subjects underlying all the work that was developed,followed by three chapters of specific research, each containing an explanatory introduction, theobtainedresultsandthespecificmethodsusedforthoseexperiments.

Thefirstspecificchapterdescribestheestablishmentofthenewmethodologyandnecessaryoptimization steps, which were done in a simple system containing the small molecule capturecompound and a purified recombinant protein that are known in the literature to interact, in thiscasethecyclicAMPandproteinkinaseA.Thefollowingchapteradvancestheestablishedprotocoltobe used with a clinically relevant drug, dasatinib, in order to identify targets in more complexsamples,suchascell lysates,and implementthe initialparameters forcapturing in livingcells.Thelast of the experimental chapters explains how this novel workflow can shed some light on thehepatotoxic side-effect of phenobarbital, an old anti-epileptic drug. Finally, a summary of thecomplete research is outlined, where the achievements are discussed and future prospectspresented.

Thisresearchprojectwas integrated inaEuropeanUnionMarieCurieActions InitialTrainingNetwork – BioChemLig. Within this consortium we had regular meetings where progress reportswerepresentedandseveralscientificandsoft-skillsseminarswereoffered. Ipresentedatotalof6communications in the3-yearperiodof theproject.Additionally, Ihad theprivilege tocollaboratecloser and host at caprotec two fellow members of BioChemLig: Valentina Bevilacqua, from thegroupofFrédéricTaranattheCEASaclayinParis,whosynthesizednewcopper-chelatingmoleculesthat were introduced in some of the capture compounds I used for my research; and MarcoBartoloni,fromthegroupofJean-LouisReymondattheUniversityofBern,whomItaughtandaidedinthecaptureoftargetsfromcelllysatesusinghisbicyclicpeptides.

The collaboration with the University of Bern resulted in a publication that I co-authored:Bartoloni,M.,Jin,X.,Marcaida,M.J.,Banha,J.etal.(2015)Bridgedbicyclicpeptidesaspotentialdrugscaffolds:synthesis,structure,proteinbindingandstability.ChemicalScience6(10):5473-5490.

DuringthecourseofthisresearchprojectIwasabletoparticipateinanEMBOPracticalCourseinChemicalBiologyfrom27Marchto2April2011attheEMBLinHeidelberg,Germanyandattendthe3rdEuropeanChemicalBiologySymposiumthattookplace1-3July2012,inVienna,Austria.

Youseethings;andyousay“Why?”ButIdreamthingsthatneverwere;andIsay“Whynot?”

GeorgeBernardShaw

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ContentsListofTables.....................................................................................................................................7ListofFigures....................................................................................................................................8Abbreviations................................................................................................................................10Summary.........................................................................................................................................11Zusammenfassung........................................................................................................................121.GeneralIntroduction..............................................................................................................131.1.ChallengesinDrugDiscovery.............................................................................................................................131.2.Drugtargetdeconvolution...................................................................................................................................15

1.2.1.Insilicoapproach.....................................................................................................................161.2.2.AffinityChromatography.........................................................................................................161.2.3.Activity-basedproteinprofiling(ABPP)...................................................................................171.2.4.Capturecompoundmassspectrometry(CCMS)......................................................................18

1.3.Clickchemistryandbio-orthogonalligations..............................................................................................211.3.1.Staudingerligation..................................................................................................................221.3.2.Copper-catalyzedazido-alkynecycloaddition(CuAAC)...........................................................231.3.3.Metal-freeclickcycloadditions................................................................................................25

2.Labelingaproteinusingclickchemistry.........................................................................282.1.Introduction................................................................................................................................................................282.2.Results...........................................................................................................................................................................31

2.2.1.Establishingandoptimizingclick-captureofPKARIusingacAMPCC.....................................312.2.2.MassSpectrometryanalysisofcapturedproteinsfromHEK293andHepG2lysatesusingaclassicalandclickablecAMPcapturecompound..............................................................................42

2.3.SpecificMethodsforthecAMP/PKARIsystem...........................................................................................482.3.1.Structuresofusedcompounds................................................................................................482.3.2.Establishingandoptimizingclick-captureofPKARIusingacAMPCC.....................................502.3.3.PreparationofHEK293lysates................................................................................................502.3.4.ClickcaptureofcAMP-bindingproteinsfromcelllysates........................................................53

3.IdentifyingDasatinibtargetsincells................................................................................543.1.Introduction................................................................................................................................................................543.2.Results...........................................................................................................................................................................58

3.2.1.Introducinganewcopper-chelatingazideintheCuAACreaction...........................................583.2.2.CapturingdasatinibtargetsinK562celllysateusingCuAAC..................................................603.2.3.CellviabilityassayfordasatinibcompoundsinK562cells......................................................683.2.4.CapturinginlivingK562cells...................................................................................................70

3.3.Specificmethodsforthedasatinibsystem....................................................................................................773.3.1.Structuresofusedcompounds................................................................................................773.3.2.OptimizingCuAACreactionparameterswithnewcopper-chelatingazide.............................783.3.3.CapturingdasatinibtargetsinK562celllysateusingCuAAC..................................................793.3.4.CellviabilityassayfordasatinibcompoundsinK562cells......................................................793.3.5.CaptureofdasatinibtargetsinlivingK562cellsusingCuAAC................................................80

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4.Phenobarbital:newbindersforanolddrug..................................................................814.1.Introduction................................................................................................................................................................814.2.Results...........................................................................................................................................................................84

4.2.1.Determiningpermeabilityofphenobarbital-alkynecapturecompounds................................844.2.2.Developingaphenotypicassaytoevaluatephenobarbitaleffect...........................................854.2.3.CapturingphenobarbitaltargetsinMH1C1celllysate.............................................................974.2.4.CaptureofphenobarbitaltargetsinlivingMH1C1cells.........................................................1014.2.5.CapturingphenobarbitaltargetsintissuewithhighEGFRexpression..................................103

4.3.Specificmethodsforthephenobarbitalsystem......................................................................................1054.3.1.Structuresofusedcompounds..............................................................................................1054.3.2.TreatmentofMH1C1cellswithphenobarbital.......................................................................1064.3.3.SubcellularfractionationofMH1C1cells................................................................................1064.3.4.TreatmentofMH1C1cellswithhighconcentrationofphenobarbital....................................1064.3.5.Treatmentofratprimaryhepatocyteswithphenobarbital..................................................1074.3.6.RT-PCR...................................................................................................................................1074.3.7.CaptureofphenobarbitaltargetsinMH1C1celllysate..........................................................1094.3.8.CaptureofphenobarbitaltargetsinlivingMH1C1cells.........................................................1104.3.9.Captureofphenobarbitaltargetsinhumanplacentalysate.................................................111

5.ConclusionsandOutlook....................................................................................................1126.GeneralMethodsandMaterials.......................................................................................1166.1.CellCulture...............................................................................................................................................................116

6.1.1.HEK293cellculture................................................................................................................1166.1.2.K562cellculture....................................................................................................................1166.1.3.MH1C1cellculture..................................................................................................................1176.1.4.Ratprimaryhepatocytesculture...........................................................................................117

6.2.BiochemistryGeneralMethods.......................................................................................................................1186.2.1.Proteinconcentrationdetermination....................................................................................1186.2.2.SDS-PAGE...............................................................................................................................1186.2.3.PolyacrylamideGelStaining..................................................................................................1196.2.4.WesternBlotting...................................................................................................................120

6.3.MassSpectrometry...............................................................................................................................................1216.3.1.Sampleacquisitionanddatabasematchidentification........................................................121

6.4.MolecularStructuresofCompounds............................................................................................................1226.4.1.Capturecompounds..............................................................................................................1226.4.2.Competitors...........................................................................................................................1256.4.3.Ligands..................................................................................................................................125

6.5.Materials...................................................................................................................................................................1266.5.1.Chemicalreagents.................................................................................................................1266.5.2.KitsandConsumables............................................................................................................1276.5.3.Buffers,mediaandsolutions.................................................................................................1286.5.4.Equipment.............................................................................................................................129

References...................................................................................................................................130

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ListofTablesTable2–1:SummaryofidentificationsfromcAMPcaptureexperimentsinHEK293andHepG2lysates............42Table2–2:ProteinsidentifiedasspecificallycompetedwithcAMPacrossthedifferentcapturemethodsineach

typeofcellularlysate.........................................................................................................................43Table2–3:NumberofproteinsidentifiedascompetedbycAMPincommonbetweenthedifferentsamples...44Table2–4:Calculatedintensityfold-changes(assayvs.competition)andMScountsforidentifiedPKAsubunits

ineachsample...................................................................................................................................45Table2–5:SummaryofidentificationsfromcAMPcaptureexperimentsinHEK293lysates...............................46Table2–6:Calculated intensity fold-changes (assayvs. competition)andcorresponding significancevalue for

identified PKA subunits from capture experiments using full and clickable cAMP capturecompounds........................................................................................................................................47

Table2–7:SummaryofparametersutilizedinPKARIlabelingandcapturingwithacAMPcapturecompoundviaaCuAACreaction...............................................................................................................................52

Table3–1:SummaryofidentificationsfromdasatinibcaptureexperimentsinK562lysate................................60Table3–2: Listof competedand significanthits indasatinib captureexperiments inK562 lysateusingapre-

clickedworkflow................................................................................................................................61Table3–3:Listofcompetedandsignificanthits inDasatinibcaptureexperiments inK562 lysateusingaclick

workflow............................................................................................................................................64Table3–4:ValuesforexpectedtargetsofDasatinibinK562lysateusingaclickworkflow.................................65Table 3–5: Gene Ontology term enrichment from significantly competed proteins in Dasatinib capture

experimentinK562lysateusingaclickworkflow.............................................................................66Table3–6:Effectofdasatiniborclickabledasatinib-alkynecapturecompoundonK562cellviability................69Table3–7:SummaryofidentificationsfromdasatinibcaptureexperimentsinK562livingcellsinnineseparate

experiments.......................................................................................................................................70Table 3–8: Excerpt of potential hits for dasatinib in K562 living cell capture using a click workflow from 9

individualexperiments......................................................................................................................72Table3–9:Top20list(sortedbyincreasingpvalue)ofdasatinib-competedproteinswhencalculatingvaluesfor

allrunsfromallexperiments.............................................................................................................73Table 3–10: Gene Ontology term enrichment from significantly competed proteins in dasatinib capture

experimentinK562livingcellsusingaclickworkflow......................................................................75Table4–1:PAMPAresultsofphenobarbitalalkynecapturecompounds.............................................................84Table4–2:SummaryofidentificationsfromphenobarbitalcaptureexperimentsinMH1C1celllysate...............98Table4–3:Potentialhits forphenobarbital inarathepatomacell line(MH1C1) from3 individualexperiments

usingaMS-basedanalysis...............................................................................................................100Table4–4:SummaryofidentificationsfromphenobarbitalcaptureexperimentsinMH1C1livingcells............101Table4–5:ListofcompetedandsignificanthitsinphenobarbitalcaptureexperimentsinMH1C1livingcells..102Table4–6:Summaryofidentificationsfromphenobarbitalcaptureexperimentinhumanplacentalysate.....103Table 4–7: List of competed and significant hits in phenobarbital capture experiments in human placenta

lysate...............................................................................................................................................103

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ListofFiguresFigure1-1:DrugResearchandDevelopmenttimeline.........................................................................................13Figure1-2:Schematicrepresentationofanaffinitychromatographypull-downprobe......................................16Figure1-3:Schematicrepresentationofanactivity-basedproteinprofiling(ABPP)probe.................................17Figure1-4:Schematicrepresentationofacapturecompound............................................................................18Figure1-5:Schematicworkflowsforthreedifferenttargetdeconvolutiontechniques.......................................19Figure1-6:Workflowforidentificationofsmall-moleculetargetsinlivingcells..................................................20Figure1-7:Illustrationofbio-orthogonalligation.................................................................................................21Figure1-8:Classical(A)andtraceless(B)Staudingerligation...............................................................................22Figure1-9:Mechanismofthecopper-catalyzedazido-alkynecycloaddition(CuAAC).........................................23Figure1-10:CuAAC-acceleratingligandsofchoice...............................................................................................25Figure1-11:Strain-promotedazido-alkynecycloaddition.ExamplereactionbetweenDIFOandanazide.........26Figure1-12:Tetrazineligation..............................................................................................................................27Figure2-1:cAMPproductionandactivationofPKA.............................................................................................29Figure2-2:LabelingofrecombinantPKARIusingCuAACinaserialdilutionofcAMP-alkynecapturecompound.

...........................................................................................................................................................32Figure2-3:LabelingofrecombinantPKARIusingCuAACinaserialdilutionofcAMP-alkynecapturecompound

withcorrespondingcompetitionsamples.........................................................................................32Figure 2-4: Effect of copper sulfate concentrationon the click labeling of recombinant PKARI using a cAMP-

alkynecapturecompound.................................................................................................................33Figure 2-5: Effect of copper ligand TBTA on the click labeling of recombinant PKARI using a cAMP-alkyne

capturecompound............................................................................................................................34Figure2-6:LabelingofrecombinantPKARIusingacAMP-alkynecapturecompoundatdifferentCuAACreaction

times..................................................................................................................................................35Figure2-7:LabelingofrecombinantPKARIusingCuAACwithdifferentbiotin-azideconcentrations..................37Figure2-8:Click-captureofPKARIusingacAMPalkynecapturecompound.......................................................38Figure2-9:LabelingofPKARIwithacAMP-alkynecapturecompoundclickedtoabiotin-azideusingCuAACwith

BTTEascopperligand........................................................................................................................40Figure2-10:EffectoftimeofCuAACreactiononthelabelingofPKARIwithacAMP-alkynecapturecompound

andaTAMRA-azide...........................................................................................................................41Figure3-1:SchematicrepresentationofthemodulardomainsoftheABLkinases.............................................55Figure3-2:Representationofauto-inhibited(closed)andactive(open)ABLkinases.........................................55Figure3-3:Proteinsinhibitedbykinaseinhibitorswithanaffinityoflessthan30nMforABL1andABL2.........56Figure 3-4: Labeling of recombinant SRC and K562 cell lysate with a clickable dasatinib-alkyne capture

compoundusinganewcopper-chelatingazidewithaTAMRAlabel................................................58Figure3-5:CCMSchartof significantlycompetedhits inDasatinibcaptureexperiment inK562 lysateusinga

pre-clickedworkflow.........................................................................................................................61Figure 3-6: STRING protein interaction analysis of significantly competed proteins in dasatinib capture

experimentinK562lysateusingthepre-clickedworkflow...............................................................62Figure 3-7: STRING protein interaction analysis of significantly competed kinases in Dasatinib capture

experimentinK562lysateusingtheclickworkflow..........................................................................64

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Figure3-8:CCMSchartofsignificantlycompetedkinasesinDasatinibcaptureinK562lysateusingCuAAC......65Figure 3-9: STRING protein interaction analysis of significantly competed proteins in dasatinib capture

experimentinK562lysateusingtheclickworkflow..........................................................................67Figure 3-10: Scheme of multi-well plate for cell viability assay of dasatinib (Dasa) and dasatinib clickable

compound(CPT392)inK562cells.....................................................................................................68Figure3-11:Luminescencedetectionofcellviabilityassayofdasatinibandclickabledasatinib-alkynecapture

compoundinK562cells.....................................................................................................................69Figure3-12:CCMSchartsofCSK,ALDOC,ABLandBCRproteinsinDasatinibcaptureexperimentinK562living

cellsusingaclickworkflow................................................................................................................74Figure 3-13: STRING protein interaction analysis of significantly competed proteins in dasatinib capture

experimentinK562lysateusingtheclickworkflow..........................................................................75Figure4-1:Luminal(phenobarbital)asitwaspackagedin1940/1945................................................................81Figure4-2:PhenobarbitalinducesCYP2BexpressionviaEGFRinhibition............................................................83Figure4-3:Synthesizedphenobarbital-alkynecapturecompounds.....................................................................84Figure4-4:WesternBlotanalysisofCARinisolatednucleiandcytosolfromMH1C1cells...................................85Figure4-5:EffectofphenobarbitalonCARtranslocationfromcytosoltonucleusinMH1C1cells.......................86Figure 4-6: Effect of phenobarbital onAMPKphosphorylation (pAMPK) inMH1C1 cells treated in serum-free

medium.............................................................................................................................................88Figure 4-7: Comparative analysis of the effect of phenobarbital on AMPK phosphorylation for each time

incubationpointinMH1C1cellstreatedinserum-freeDMEM..........................................................88Figure 4-8: Effect of serum supplementation in AMPK phosphorylation (pAMPK) from non-treated (0 µM

phenobarbital)MH1C1cells...............................................................................................................89Figure4-9:EffectofphenobarbitalonAMPactivatedproteinkinase(AMPK)phosphorylation(pAMPK)inarat

hepatomacelllinetreatedincompletemedium..............................................................................90Figure 4-10: Comparative analysis of the effect of phenobarbital on AMPK phosphorylation for each time

incubationpointinMH1C1cellstreatedinFBS-supplementedDMEM.............................................90Figure4-11:EffectofphenobarbitalinCYP2B1/2detectioninarathepatomacellline......................................91Figure4-12:EffectofhighconcentrationphenobarbitalonthedetectionofCYP2B1/2inMH1C1cellsusingrat

livermitochondriamatrixfractionaspositivecontrolforantibodyreactionquality.......................92Figure 4-13: Effect of high concentration phenobarbital on the detection of CYP2B1/2 and AMPK

phosphorylationinarathepatomacellline......................................................................................92Figure4-14:DetectionofD-aminoacidoxidase(DAO)inpurifiedfractionsandMH1C1lysate...........................93Figure 4-15: Effect of phenobarbital in the detection of D-amino acid oxidase (DAO), CYP2B1/2 and AMPK

phosphorylation(pAMPK)fromratprimaryhepatocytes.................................................................94Figure4-16:AgarosegelelectrophoresisofRNA,cDNAandPCRproducts..........................................................95Figure4-17:EffectofphenobarbitalonCYP2B1mRNAexpressioninarathepatomacellline...........................96Figure4-18:EffectofphenobarbitalonCYP2B1mRNAexpressioninratprimaryhepatocytes..........................96Figure 4-19: STRINGprotein interaction analysis of significantly competed proteins in phenobarbital capture

experimentsinMH1C1lysate.............................................................................................................99Figure 4-20: STRINGprotein interaction analysis of significantly competed proteins in phenobarbital capture

experimentsinhumanplacentalysate............................................................................................104

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Abbreviations

(p)AMPK (phosphorylated)AMP-activatedkinase IR Infrared

AKAP A-kinaseanchoringprotein LC Liquidchromatography

ATP Adenosinetri-phosphate LTQ Lineartrapquadrupole

BCA Bicinchoninicacid MS Massspectrometry

BSA Bovineserumalbumin nLC Nanoflowliquidchromatography

BTTE Bis(tert-butyltriazoly)ethanol PAGE Polyacrylamidegelelectrophoresis

cAMP Cyclicadenosinemono-phosphate PB Phenobarbital

CAR Contitutiveandrostranereceptor PBREM Phenobarbitalresponsiveenhancermodule

CC Capturecompound PBS Phosphate-bufferedsaline

CCMS Capturecompoundmassspectrometry PCR Polymerasechainreaction

CID Collision-induceddissociation PI Proteaseinhibitorcocktail

CuAAC Copper-catalyzedazido-alkynecycloaddition

PKA ProteinkinaseA

CYP CytochromeP450 RNA Ribonucleicacid

DDM n-Dodecylβ-D-maltoside RP Reversephase

DMEM Dulbecco’smodifiedEaglemedium SA-MB Streptavidin-coatedmagneticbeads

DMSO Dimethylsulfoxide SDS Sodiumdodecylsulfate

DNA Deoxyribonucleicacid TAMRA Tetramethylrodamine

DTT Dithiotreitol TBS Tris-bufferedsaline

ECL Enhancedchemiluminescence TBS-T Tris-bufferedsalinewith0.1%(v/v)Tween

EDTA Ethylene-diamine-tetra-aceticacid TBTA Tris(benzyltriazolylmethyl)amine

EGFR Epidermalgrowthfactorreceptor THPTA Tris(3-hydroxypropyltriazolylmethyl)amine

FBS Fetalbovineserum UV Ultraviolet

HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonicacid

v/v Volumepervolume

HRP Horseradishperoxidase w/v Weightpervolume

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SummaryThedevelopmentofnoveldrugtargetdeconvolutionmethodologiesisagrowingneedinthe

pharmaceuticalindustry.Theincreasedregulatoryscrutinydemandsthatbetterandsafermedicinesreachtheconsumermarketand,forthat,acomprehensivecharacterizationofthemultipletargetsandoff-targetsofthedrugearlyinthedevelopmentprocessisakeysuccessfactor.

Chemicalproteomicsadvanceshaveprovidedthetoolstostudydrugmechanismsofactioninamoredirectandunbiasedwaythantarget-basedscreeningstrategies,byanalyzing theeffects inthecontextofthefullproteomefromthetargetcellortissueofinterest.

Capturecompoundmassspectrometry (CCMS) isa robustandversatilechemicalproteomicstechniqueduetotheoriginaldesignoftheprobes.Capturecompoundsaretrifunctionalmoleculesthattraditionallycarryaselectivityfunction,consistingofthesmallmoleculeunderstudy,attachedtoascaffoldbearingatag,suchasafluorophoreorbiotin,andaUV-reactivemoietyprotrudingfromthecentralcorethatcovalentlybindstheprobetothetargetprotein.Althoughcapturecompoundsareveryefficientandspecificintargetingofevenlowaffinityandlowabundanttargets,theirusehasbeenlimitedsofartoworkingwithcellularlysatesortargetingcell-surfaceproteins.

ThemainobjectiveofthisworkwastoadvanceCCMStoimplementandoptimizeworkflowsfortheuseofnewlydesignedpermeablecapturecompoundstoprofilesmallmoleculedrugsinlivingcells,wherethetargetsareintheirunalterednaturalenvironment.Bio-orthogonal“click”chemistryprovidestheprerequisitefortheuseofpermeablecapturecompoundsbearingonlytheselectivityandreactivityfunctions,andasmall“clickable”chemicalhandletobindandcross-linkthetargetsinlivingcellsandafterwardsspecificallyattachthesorting/detectionfunctionbyacontrolledchemicalclickreactionsuchasthecopper-assistedazide-alkynecycloaddition(CuAAC).

Initial reactionparameters for theclickreactionweretestedusingaclickablecAMPcapturecompoundinasolutionofrecombinantPKARI.Theoptimizationeffortsincopperionconcentration,ligandnature,durationandpHofthereaction,azideandalkyneconcentrationsyieldedsubstantialimprovementsfromthestartingpoint.CapturingPKAsubunitsfromcell lysateswiththeoptimizedparametersprovedtobepossibletousethisworkflowbuttheresultswerenotfullysatisfactory.

Acapturecompoundwithanon-naturalselectivityfunctionwasusedtofurtherdeveloptargetidentificationincelllysatesandlivingcellsusingthenewclickreaction.Thekinaseinhibitordasatinibwasprofiledusingthisstrategyandbonafidetargetsweresuccessfullyidentifiedusingtheclickablecompound. ABL, BCR and SRC were specifically identified in lysates and CSK, a known dasatinibbinder,waspositivelyidentifiedforthefirsttimebychemicalproteomicsinlivingcells.

Thenewlyestablishedclickworkflowallowedtobettercharacterizephenobarbital,adrugthatwas developed as an anti-epileptic but elicits unexplained hepatocarcinogenic effects in somespecies. The resultsof captureexperiments suggest that this smallmolecule interactswith severalproteinsintheepidermalgrowthfactorreceptorsignalingpathwayandthattheprimarytargetcouldbethetranscriptionfactorYBX1,andnotthereceptoritselfasitwassuggestedrecently.Acell-basedassaywasdevelopedtovalidatethisfindingbutwasnotfurtherexploredduetotimeconstraints.

In summary, the strategy to identify small molecule targets in living cells using capturecompoundsandbio-orthogonalchemo-selectiveligationmethodswassuccessfullyestablished.Thismethodprovides the tools forabettercharacterizationof the targetprofilesofdrugsandstillhaspotentialtobefurtherinnovated.

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ZusammenfassungDie Entwicklung neuer Methoden zur Dekonvolution der Profile von Zielproteinen für

pharmazeutische Wirkstoffe ist von wachsender Bedeutung für die Pharmaindustrie. Eineumfassende Charakterisierung der Zielproteine einschließlich sogenannter „Off-targets“ ist einSchlüsselfaktorumbessereundsicherereMedikamenteaufdenMarktzubringen.

Fortschritte in der Chemischen Proteomanalyse bieten Werkzeuge für die direktere undbezüglich der Zielproteine hypothese-freie Untersuchung der Wirkungsmechanismen vonMedikamenten, bei der die Bindung imKontext des vollständigen Proteomsder Zielzelle oder desZielgewebesuntersuchtwird.

Die Capture CompoundMassenspektrometrie (CCMS)-Technologie ist eine robuste und sehrflexibel einsetzbare Technik in der chemischen Proteomanalyse. Capture Compounds sindtrifunktionelleMoleküle,dieinihrerklassischenVersioneineSelektivitätsfunktiontragen,diedemzuuntersuchenden Kleinmolekül-Wirkstoff entspricht, die mit einer Gerüststruktur verbunden ist,welche außerdem eine Isolierungsfunktion sowie eine UV-aktivierbare Reaktivitätsfunktion für diekovalente Verbrückung zum gebundenen Zielprotein enthält. Obwohl Capture Compounds sehreffizientundspezifischZielproteinebindet,warihrEinsatzbislangwegenfehlenderPermeabilitätaufZell-Lysatebzw.aufOberflächenproteinelebenderZellenbeschränkt.

Ziel der vorliegenden Arbeit war die Weiterentwicklung der CCMS-Technologie durchImplementierung und Optimierung des Einsatzes permeabler Capture Compounds neuartigenDesignszurProfilierungvonKleinmolekül-Wirkstoffen in lebendenZellen, indenendieZielproteinein ihrernatürlichenUmgebungvorliegen.Bio-orthogonale „Click“-ChemiebietetdieVoraussetzungfür den Einsatz permeabler Capture Compounds, die lediglich die Selektivitätsfunktion, dieReaktivitätsfunktion sowie eine kleine, „Click“-fähige chemische Gruppe enthalten. Bindung undVerbrückungzudenZielproteinenkannindenlebendenZellenerfolgen,understdanachdieSortier-/Detektionsfunktion in einer kontrollierten chemischen „Click“-Reaktion (wie z.B. die Kupfer-katalysierteAzid-Alkin-Cycloaddition,CuAAC)angebrachtwerden.

Initiale Reaktionsbedingungen für die „Click“-Reaktionwurdenmit einer Capture Compoundmit cAMP als Selektivitätsfunktion in einer Lösung einer rekombinanten PKARI getestet. DieOptimierung der Reaktion brachten substantielle Verbesserungen in der Reaktionsausbeute imVergleichzudenStartbedingungen.

Um die Identifizierung von Zielproteinen in Zell-Lysaten und lebenden Zellen unterVerwendung der „Click“-Reaktion weiter zu entwickeln, wurde eine Capture Compoundmit eineranderen Selektivitätsfunktion verwendet. Das Kinaseinhibitor-Medikament Dasatinib wurde mitdieser Strategie profiliert. Diebona fide Zielproteine ABL, BCR, und SRCwurden spezifisch in Zell-LysatenundzumerstenMalCSKmitchemischerProteomanalyseinlebendenZellenidentifiziert.

Der neu etablierte methodische Arbeitsablauf ermöglichte es, Phenobarbital besser zucharakterisieren, weil es in einigen Spezies hepatocarcinogen wirkt. Die Ergebnisse der Capture-Experimentelegennahe,dassderprimäreBinderderTranskriptionsfaktorYBX1seinkönnte.

InsgesamtkonntedieStrategie,ZielproteinevonKleinmolekül-WirkstoffeninlebendenZellenmitHilfeneuartigerCaptureCompoundsundbio-orthogonalerchemo-selektiverLigationerfolgreichetabliert werden. Diese Methode bietet ein Werkzeug für die bessere Charakterisierung derZielprotein-ProfilevonMedikamentenundhatdasPotential,nochweiterverbessertzuwerden.

Chapter1:GeneralIntroduction

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1. GeneralIntroduction1.1. CHALLENGESINDRUGDISCOVERY

Thepharmaceutical industry is facingunprecedented challenges to its businessmodel.Overthepastdecade,concernsabouttheindustry’sintegrityandtransparencyregardingdrugsafetyandefficacyhavebeenraised,andleadtoanincreasedregulatoryscrutiny1.

The traditional drug development timeline (Figure 1-1) consists of a discovery phase,whichincludesthetarget-to-hit,screening,hit-to-lead,andleadoptimizationstagesthatprovideaclinicalcandidate for the development phase consisting of pre-clinical, phases I, II and III clinical trials,culminatinginasubmissiontothecompetentauthoritiesforadruglaunch.

Figure1-1:DrugResearchandDevelopmenttimeline.


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In the initial stageof thediscovery phase a target is selected either by actively pursuing anunmetmedicalneedorbyscreeningliteraturewithreportsonnovelassociationswithdiseasestates,always guided by the size of the potential market. In the target-to-hit phase it is important todetermine the differences and similarities across species for future safety studies translation.Following the target identification, a screening for potential hits is performed using novel highthroughput technologies scanning small molecules or biologics, such as antibodies or RNA/DNA-based compounds. Opposite to the large-scale profiling, amore direct approach in drug-design issometimes utilized via computational molecular modeling prediction systems. Once a potentialcandidate is identified, a series of analogues are synthesized and structure-activity relationships(SAR) are measured as well as initial pharmacokinetics (PK) studies are performed. In the leadoptimization stage, thepotential drug is refined and improved for efficacy andpotency aswell asidentification of undesired off-targets. At this stage a drug candidate can enter the developmentphase and initiate the pre-clinical studies, which include drug formulations compatible with safeadministration, further PK studies andADME (Absorption,Distribution,MetabolismandExcretion)analysis in disease models. Safety and toxicity studies are focused on unwanted effects incytochromeP450(CYP)metabolizingenzymes,orspecific ionchannelsthatcan leadtodeleteriousconsequences.With safety and toxicity parameters in check, the phase I clinical trial consists of asmall groupof individuals,usuallyhealthyvolunteers,wheredosagesand frequencyare tested. InPhase II the cohort is increased and the effectiveness of the compound is provedbydoubleblindstudies,whichcontinuesontoPhaseIIIwithmoresubjectsandalsodiversifiedgeneticbackgroundstodetermineobservedsideeffectsandestablishthegeneralbenefitsofthenewmolecularentityincomparisontothecurrentstandardofcare.

AsFigure1-1shows,navigatinganewmedicinetothemarketisalongandstrenuouspath.Alotofpotentialdrugsdonotmoveforwardinthepipelinebecauseofundesiredeffectsorproblemsofformulation.Astudyonthesuccessratesforinvestigationaldrugs2covering835drugdevelopers,includingbiotechcompaniesaswellasspecialtyandlargepharmaceuticalfirmsfrom2003to2011,reportsthatfromthe7.300independentdrugdevelopmentpathsanalyzed,asmuchas10.4%ofallindication development paths in phase Iwere approved by theUS Food andDrug Administration(FDA).Thebiggesthurdlealong thedevelopmentchain is the transition fromphase II tophase III,with only 32.4% of the drugs advancing in the pipeline, making companies carefully analyze therisk/costbenefitsoftheseundertakings.Hayetal.2suggestonewaytoimprovethesuccessratesisto improve the predictive animal models, perform earlier toxicology evaluation, biomarkeridentificationandnewtargeteddeliverytechnologiesthatmayincreasefuturesuccessintheclinic.


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1.2. DRUGTARGETDECONVOLUTIONOne way to improve the success rates in the drug development pipeline, discussed in the

previousparagraph,istoimplementtechniquesofdrugtargetdeconvolutionearlyonintheprocess.Drug target deconvolution is a process where the action of a drug, a small molecule, is

characterized by identifying the proteins binding the drug and initiating the biological effect. Thebiological relevant targethas tobeextracted,ordeconvoluted, froma listofproteins identified insuch an approach. Besides the medically desired action of the drug, the identification of otherproteinsbindingthedrugcanhelpto identifysideeffectsandtoxicityataveryearlystageofdrugdevelopment3.

Traditionally,newdrugshavebeendeveloped followinga target-based screening inwhichalargelibraryofcompoundsisscreenedagainstasingletargetproteinthoughttoberesponsibleforthedisease,andsubsequentlytheactivehitscanbeoptimizedthroughmedicinalchemistryefforts.Onemajor limitationof thisapproach is the fact thatmanycompoundsare found to interactwithmultiple targets. The “one drug, one target” paradigm, thought to be the cornerstone of target-basedmethods,oftendoesnotholdtrueforcompoundsidentifiedusingthisstrategy4.

Oneofthemajoradvantagesofphenotype-basedapproachesisthattheyprovideanunbiasedway to find active compounds in the context of complex biological systems. Because phenotypicscreening takes place in a physiologically relevant environment of cells or whole organism, theresults fromsuchscreensprovideamoredirectviewof thedesired responsesaswellashighlightpotentialsideeffects.Moreimportantly,phenotypicscreenscanleadtotheidentificationofmultipleproteinsorpathwaysthatmaynothavebeenpreviouslylinkedtoagivenbiologicaloutput.Becausetargetidentificationfromphenotypicscreensisexpectedtogenerateaspectrumofpossibletargets,theterm“targetdeconvolution”wascoinedtomoreaccuratelydefinetheprocess4.

Recent developments in chemical proteomics, a multidisciplinary research area integratingbiochemistryandcellbiologywithorganic synthesisandmass spectrometry,haveenabledamoredirect and unbiased analysis of a drug’s mechanism of action in the context of the proteome asexpressedinthetargetcellorthetissueofinterest.Advancesinquantitativemassspectrometrynowenablethedetectionandquantificationofdrug-inducedchangesinproteinexpressionandactivationwith unprecedented proteome coverage. There aremultiple approaches that can be employed inchemical proteomic workflows. These include small molecule affinity-based and activity-basedprobes that canbeused to isolate targets andmore recently, in silico techniques topredict smallmoleculebindingproteins.Moreover,affinityenrichmentstrategiesusingimmobilizeddrugsortoolcompounds now allow to characterize the expressed “interactome” of a drug directly from cellextracts5.

Indrugdevelopment,chemicalproteomicscanshedanunbiasedlightontheinteractionofthedrugmoleculewiththeproteomeandidentifybothmoleculartargetsresponsibleforbenigneffectsand unwanted potentially toxic interactions. Furthermore, a different attachment position of thedrug to the rest of the probe results in different interaction profiles with the proteome. Thisfavorable “sideeffect”of usingmulti-functional probesmaygive insight into the structure activityrelationship(SAR)ofthedrugandmayserveasstartingpointforchemicalmodifications6.


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1.2.1. InsilicoapproachComputeraideddrugdesign isamajorpillarof target-baseddrugdiscovery.Onthebasisof

docking studiesandvirtual screening,drugcandidateswithoptimalpotencyandselectivity canbepredicted. With the recent renaissance of phenotype-based drug discovery, these in silicotechnologieshavefoundanimportantnewroleintheprocessoftargetprediction.Overthelasttwodecades,extensive informationregardingactivity,structuresandtargetsofsmallmolecule librarieshas beendeposited into public databases such as ChEMBL,DrugBank andChemBank.Using thesetools, targetsof active compounds canbepredictedbasedon similarities in structurebetweenanactivehitandwell-characterizeddrugsinthesedatabases.Thiscomputeraidedtargetpredictionhasbeenwidelyusedtoidentifynewtargetsofknowndrugs,topredictthetargetsofactivehitsfromalibrary screening, and to investigate themechanism of action of hits discovered from phenotypicscreens4.

Thepreviouslydescribedstrategyreliesonmoleculesthathavealreadybeensynthesizedandstudied inaspecific system, fromwhichresultsareextrapolated foranewprotein/small-moleculepair. The drug-like chemical space, comprising organic small molecules (MW < 500 Da) ofintermediate polarity, is very large andmight contain on the order of 1060 possiblemolecules.Denovo drug design consists in exploring this chemical space in search for bioactive compounds bygeneratingandscoringvirtualmoleculespriortotheirsynthesis,wherebyscoringreliesonavarietyofvirtualscreeningapproachessuchasligand-basedandtarget-basedmethods.Theadvantageofdenovodrugdesignisthatmanymoremoleculescanbeevaluatedthanwhatispossibleifconsideringonly already synthesized molecules, a strategy which might help to uncover lead structures andeventuallyimproveclinicalsuccessfornewdrugs7.

1.2.2. AffinityChromatographyAffinitypurificationisthemostwidelyusedtechniquetoisolatespecifictargetproteinsfroma

complex proteome. Smallmolecules identified in phenotypic screens are immobilized onto a solidsupportthatcanbeusedtoisolateboundproteintargets(Figure1-2). The small molecules used as selectivity functions in thesestudies display binding to their target proteins with lownanomolar affinity. This approach relies on extensive washingsteps to remove non-binders, followed by specific methods toelutetheproteinsofinterest.Theelutedproteinscantheneitherbe directly identified using ‘shotgun’ type sequencing methodswith multidimensional liquid chromatography or be furtherseparated by gel electrophoresis and analyzed by massspectrometry.Theidentifiedpeptidesequencescanthenbeusedindatabasesearchestoidentifythetargetprotein4.

Representativestudiesutilizingthistechniqueproducedso-called kinobeads to study the kinase subproteome from eukaryotic cell lines. These beads carry anumberofdifferent smallmoleculekinase inhibitorsandhavenotonlybeenused tomapkinasesfromcomplexbiologicalsamples,butalsotoassesstheselectivityprofilesofkinaseinhibitordrugs8.Daubandcolleaguesusedadifferentsetofkinaseinhibitorstoisolatelargenumbersofkinasesfrom

Figure1-2: Schematic representationofanaffinitychromatographypull-downprobe.Bi-functional molecule bearing atargeting/selectivity function (redrectangle), corresponding to the smallmolecule under study, and a sortingfunction to allow attachment to a solidmatrix,usuallyviabiotin/avidininteraction.ReproducedfromLenzetal.6


17

celllysates,andalsofocusedonthesubsequentanalysisofthephosphorylationstateoftheenrichedproteins9.

Themajor problemof affinity chromatography and its “pull-down” approach consists in theevaluation of nonspecific binding especially if we take into consideration that some intracellularproteins (e.g. glyceraldehyde-3-phosphate dehydrogenase) are detected as components of varioussubproteomes.Itisalsopossiblethatroughelutionconditionsmayremoveproteintargetsexhibitingmoderateaffinitytoligandsused.Affinity-basedapproachesarealsoapplicableforidentificationofsmall-molecule protein targets, but the previously described limitations demand a subsequentanalysisofidentifiedproteinswithaparticulartargetinhibitionassay10andaremostlyunusableinlivingcells.

1.2.3. Activity-basedproteinprofiling(ABPP)Activity-based probes are small molecule tools that can be used to monitor the activity of

specificclassesofenzymes.ABPPprobesarecharacterizedbyaselectivityfunctionthatiscapableofcovalentlyreactingwiththeactivesitesoftargetenzymesdrivenbythecatalyticmechanismoftheenzyme. By directly modifying the active site amino acid residues, ABPP probes act as suicideinhibitors. Over the past decade and a half, various ABPP probes have been designed to studyproteases,hydrolases,phosphatases,histonedeacetylases,andglycosidases,andtheseprobeshaveproven to be valuable in investigating enzyme-related disease mechanisms including cancer andmetabolic disorders. Typical activity-based protein profiling follows a similar overall workflow asaffinity chromatography, including probe binding, protein separation, sequence analysis, anddatabasesearching.However,becausemostABPPprobesaredesignedtotargetaspecificenzymeclass, they aremostly useful for the development of small-moleculeswhere a specific enzyme orenzymefamilyissuspectedtobeinvolvedinacertaindiseasestateorpathway4,6.

ABPP probes have three components: a reactiveelectrophile for covalent modification of enzyme active site, alinker or a specificity group for directing probes to specificenzymes,andareporteroratagforseparatinglabeledenzymes(Figure1-3).CovalentenzymeinhibitorscanbereadilyconvertedtoABPPprobesbyattachinga tagginggroup,and thecombineduse of covalent inhibitors and ABPP probes in phenotypicscreeninggreatlyfacilitatestargetidentificationandalsooffersapowerful tool to study function and mechanism of identifiedproteins. In order to covalently attach ABPP probes to targetproteins, an active sitenucleophile suchas cysteineor serine isrequired4,6.

ABPP probes are not only useful for target identification,butalsoarepowerfultoolsforthediscoveryofdiseaserelatedproteinsandthetechnologyhasbeenusedtoidentifyserineandcysteinehydrolasesfromhumancancersamplesorproteolyticenzymesof the proteasome, for example.Mechanism-based ABPP-probes have also been reported for theprofiling of native ATP-binding proteins, in particular kinases. These probes exploit the catalyticreaction of the kinase to form a chemical group that can covalently target lysine residues in the

Figure 1-3: Schematic representation of anactivity-basedproteinprofiling(ABPP)probe.Typically a bi-functional probe where thesmallmolecule as selectivity function reactswiththeactivesiteofthetargetproteinandestablishes a covalent bond. The attachedsorting function can either be a reporter,such as a fluorophore for visualization, or atag, such as biotin for isolation andenrichment.ReproducedfromLenzetal.6


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catalytic sites. Othermechanism-based ABPP-probeswere successfully applied for the profiling oftyrosine phosphatases, phospholipases, glycosidases, cytochrome P450 proteins, and the list isgrowingatafastpace6.

ThelimitationofABPPprobesinpharmaceuticaltargetresearchisthatmostsmall-moleculesarenon-covalentbindersandmodifying theselectivity functionwhere it interactswith theproteinwould most probably alter its binding affinity. A new design of probe was developed where thesmall-moleculeinteractswiththetargetproteininanaturalreversiblemanner,butanindependentphoto-reactivemoiety forms a covalent ligation to the target allowing for the necessary stringentwashingstepstoreduceunspecificbinders–theCaptureCompound11.

1.2.4. Capturecompoundmassspectrometry(CCMS)Smallmoleculeprobes termedCaptureCompoundsare tri-functionalmoleculescontaininga

selectivityfunction,aphotoactivatablereactivityfunction,andasortingfunctionprotrudingfromacommoncoreatom(Figure1-4).Bindingoftheselectivityfunctionoccursnon-covalentlydrivenbythe thermodynamic equilibrium. Covalent cross-linking to the targetprotein isaccomplished throughphoto-activationofaseparatereactivityfunctionthat,incontrasttoclassicphotoaffinitylabeling,isnotpartoftheselectivityfunction,butislocatedonadifferentbranch of the probe, intentionally at a certaindistance from the selectivity function 11. As a result,all three functional entities exhibit a high degree offreedomindependentofeachother.Inparticular,dueto the linker between selectivity function and coreatom of the probe, the selectivity function binds tothe small molecule binding pocket of the targetproteins, whereas the reactivity function stays at the protein surface. Therefore, the reactivityfunctioncantargetsurface-exposedstructuresofthetargetproteins,andisnotstructurallyconfinedwithinthesmallmoleculebindingpocketoftheprotein.Thisflexibilityofthephoto-reactivefunctionallowsforhighcross-linkyields12.

In a typical CCMS experiment, the selectivity function accomplishes an equilibrium-drivenaffinity binding to target proteins. In a second step, this equilibrium is “frozen” through photo-activationof the reactivity function. This results in a covalentbond formationbetweenprobeandproteintargetthroughthereactivityfunction.Becausetheequilibriumbindingofthetargetproteinsis“frozen”bythecovalentcross-linkofthereactivityfunctiontotheprotein,weak,butstillspecificbindersareretainedattachedtotheprobeduringsubsequent isolationandwashingstepsandcanbe identified. The thirdmodule is a sorting function for isolation of the targeted proteins, usuallybiotin that can be enriched with streptavidin-coated magnetic beads for subsequent proteomicanalysis.ThebasicexperimentalworkflowofCCMSandthepreviouslydescribedaffinitypull-downandABPPareillustratedinFigure1-5,onpage19.

Capture compounds with different selectivity functions have been used for the isolation ofprotein familiessuchaskinases 13,14,GTPases 15,cAMP-bindingproteins 12,methyltransferases 16,17,

Figure1-4:Schematicrepresentationofacapturecompound.Tri-functional molecule consisting of a selectivity function,formed by the small molecule under study, a reactivityfunction that establishes a covalent bond with the targetproteinuponUV irradiation,anda sorting function that canbeafluorophoreforvisualizationoratag,suchasbiotin,forenrichmentandisolation.ReproducedfromLenzetal.6


19

and 2-oxoglutarate oxygenases 18 from complex biologicalmixtures. In each case, specific binderswereisolatedandidentifiedfromcell lysatesorsubcellularfractionsoftissues.Ineachofthecitedcapture compound studies, the yield of identified specific binders was shown to be substantiallyhigher than inparallelaffinitypull-downexperimentswithoutphoto-activatedcross-linkingvia therespectivereactivity functions6.Particularly inthecaseofmethyltransferasesthatwereaddressedbyacapturecompoundwiththemethyltransferaseproductinhibitorS-adenosyl-L-homocysteineasselectivityfunction,theresultsdemonstratedthatacapturecompoundbasedapproach,duetotheadditionalphoto-activatedcross-linktotheboundtargetproteinsviathereactivityfunction,candealwithsmallmolecule-protein interactions inarangeofrelatively lowaffinity, i.e., inthemicromolarrange.Theseinteractionsareusuallydifficulttoaddressbysimpleaffinitypull-down.

Figure1-5:Schematicworkflowsforthreedifferenttargetdeconvolutiontechniques.In affinity chromatography pull-down experiments (panel A), probes reversibly interact with target proteins defined by a dynamicequilibrium.Non-bindingproteinsarewashedaway,andretainedproteinsareanalyzedafterelutionusingdenaturingbufferoranexcessoffreeaffinity ligand.Thechoiceofbufferconditionsrequiresatradeoff: lowstringencywill leadtohighunspecificbackgroundbinding,while high stringency will lead to loss of weakly, but still specifically interacting proteins. In activity-based protein profiling (ABPP)experiments (panelB), the selectivity functionof theprobebinds toand chemically reactswith theactive siteof targetenzymes.Harshwashingconditionscanbeappliedtoremoveproteinsnonspecificallyboundtothesolidphase.Asalimitation,ABPPcanonlybeusedwithsmall molecules that chemically react with their targets. In Capture Compound Mass Spectrometry (CCMS) experiments (panel C) theCaptureCompoundselectivity function reversibly interactswith targetproteinsdefinedbyadynamicequilibrium,and thisequilibrium isfixed through a photo-induced cross-linking of the reactivity function. Harsh washing conditions can be applied to remove unspecificbackground.Somenonspecificallycross-linkedproteinsareretainedaswell.ReproducedfromLenzetal.6


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Theprerequisiteforthesuccessfulfunctional isolationofproteinsfrombiologicalsamplesbysmall molecule probes is that the proteins are in their native (functional) state. Some proteinstoleratetransfer fromthecellularcontext toacellular lysate,whileothersmaynot. It is thereforeimportant to keep the proteins in a state as native as possible in order to reveal physiologicallyrelevantsmallmolecule–proteininteractions.

Thevastmajorityofchemicalproteomicsstudieshavebeenconductedonlysatesamples16,19–21. Ideally, probing of small molecule–protein interactions on intracellular proteins should beconductedinintactcellswithmembrane-permeableprobes.Thepermeabilityoftheprobescanbeincreasedbyonlyaddingthetagafterthesmallmoleculehasfoundthetargetandisfixedtoit.Thisis achieved using chemo-selective ligatable probes. The smaller probes comprise only a combinedselectivity/reactivityfunction(inABPPprobes)orseparateselectivityandphoto-reactivityfunctions(in capture compounds), but no sorting or detection function. Instead, the probes contain a smallfunctional group, such as an alkyne or azidemoiety. This enables chemical ligation of the sortingfunction later in theworkflow. The sorting function is attached to the probeonly after the targetrecognition and (photo-) cross-linking reaction has taken place and cell integrity is no longernecessary. This reaction workflow is called chemo-selective bio-orthogonal chemical ligation.Permeable probes consisting only of selectivity and reactivity function or combinedselectivity/reactivity functions have been successfully used to address targets of small moleculeswithinlivingcells22–24.

The in-cell workflow for the application of either a chemically reactive or a photo-reactiveprobeisdepictedinFigure1-6.

Figure 1-6: Workflow for identification of small-moleculetargetsinlivingcells.Permeable small molecule probes carry aselectivity function and a reactivity function or acombinedselectivity/reactivityfunction,butlackasortingfunction.Instead,theprobecarriesasmallfunctionalgroupthatenablesadditionofasortingfunction througha specific ligation reaction laterin the workflow. Chemical or photoactivatedcross-link can be carried out, and only after thatthecellislysedforligationtothesortingfunction.ReproducedfromLenzetal.6

Anessentialconditionforthis

approach is that the functions onthe small probe and the sortingfunctiononly reactwitheachotherand not to any other intracellularcomponent.

In essence, it needs to allowforabio-orthogonalchemoselectiveligation.


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1.3. CLICKCHEMISTRYANDBIO-ORTHOGONALLIGATIONSIn the advent of successfully using green fluorescent protein (GFP) tags to visualize protein

dynamicsandfunctioninlivingsystems,bycleverlyintroducingagenethatconcomitantlytranslateswith the target protein and produces an active fusion protein, it quickly became clear that thisstrategydidnotallowtostudypost-translationalmodificationsorotherrelevantbiologicalmoleculessuchasglycans, lipids,metabolitesandnucleicacids 25,26.Toovercomethis limitation,a reactivity-basedbio-orthogonalchemistryapproachwassuccessfullydeveloped.

Bio-orthogonal reactant pairs, which aremostsuitableforsuchapplications,aremoleculargroupswith the followingproperties: (i) theyaremutually reactive but do not cross-react orinteract in noticeable ways with biologicalfunctionalities or reactions in a cell, (ii) they andtheir products are stable and non-toxic inphysiological settings, and (iii) ideally, theirreaction is highly specific and fast 25,27. Anillustration from Sletten et al. 26 depicts thechallenge of “fishing” out a specific molecularentity froma “sea”of functional groups thatarepresentincells(Figure1-7).

Bio-orthogonal chemical reactions haveemergedashighlyspecifictoolsthatcanbeusedfor investigating the dynamics and function ofbiomolecules in living systems 27–29. Clickchemistry,inspiredbynature’suseofsimpleandpowerfulconnectingreactions,describesthemostspecificbio-orthogonalreactionsthatarewideinscope,easytoperform,andusuallyemployreadilyavailablereagentsthatareinsensitivetooxygenandwater30–32.Invivobio-orthogonalchemistryandclick chemistry therefore overlap quite a bit, reflecting the same underlying chemical principlesappliedinsomewhatdifferentwaystowardthediscoveryordevelopmentofmolecularfunctionandinformation25.

With over 20 bio-orthogonal transformations now reported in the literature and new onesbeing discovered at a rapid pace, selecting the “best fit” for a given application is non-trivial. Thechemistries vary widely in terms of their selectivities and biocompatibilities, and many of theirperceivedstrengthsandweaknessesremainanecdotal29.

Oneofthemostpopularbio-orthogonalfunctionalgroupsistheazide.Thesmallsize,coupledwithitsinertnaturetowardsendogenousbiologicalfunctionalities,setstheazideapart.Additionallytheazidehasuniquechemistrieswithotherbio-orthogonalfunctionalities,mostnotablyphosphinesand alkynes 28. Because of this versatility, old andnewlymodified reactions basedon azides havebeenthebasisfortheexpansionofclickchemistryinthebiologicalcontext,suchastheStaudingerligation,[3+2]cycloadditionswithorwithoutmetalcatalystsand,morerecently,atetrazineligation.

Figure1-7:Illustrationofbio-orthogonalligation.The bio-orthogonal reactant pairs should react exclusively witheachotherformingastableandnon-toxicproducttothecellularenvironment.ReproducedfromSlettenetal.26


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1.3.1. StaudingerligationTheStaudingerligationessentiallylaunchedthefieldofbio-orthogonalchemistry,notbecause

itwas the first bio-orthogonal reaction per se, but because itwas the first among entirely abioticfunctionalgroupsandthereforehadthepotential fortranslationto liveorganisms27. Itsprototypereaction was the Staudinger reduction of azides with triphenylphosphine and water, reported byHermann Staudinger in 1919. It appeared well suited as a prototype for bio-orthogonal reactiondevelopment because the two participantswere abiotic,mutually and selectively reactive,mostlyunreactivewithbiologicalfunctionalities,andtolerantofwater.In2000,SaxonandBertozzi33wereabletoadaptthisreaction intoa ligationmethodwheretheysuccessfully labeledmodifiedglycansonthesurfaceofcellsviathenewlydevelopedStaudingerligation(Figure1-8,panelA).

Figure1-8:Classical(A)andtraceless(B)Staudingerligation

The Staudinger ligation was promising but there were some limitations. The phosphinereagentsslowlyunderwentairoxidationwithinbiologicalsystemsandwereprobablymetabolizedbycytochromeP450enzymes inmice.Additionally, the kinetics of the reactionwasnot fast enough,whichnecessitatedtheuseofhighconcentrationsofphosphinereagent.

Bertozzi’s groupovercame this limitation later in the same yearby developing a “traceless”Staudingerligation(Figure1-8,panelB)wherethepositionofthereportermoduleinthephosphinereagentwasmodified.Thisallowedthephosphineoxidetobeexpelledduringthereaction leavingthetwogroupslinkedviaanamidebond.

Sincethistime,themechanismofthereactionandotherdetailshavebeenprobedindepth34–36, andanumberof iterationshavebeen reported, suchas fluorogenic versions, includinga FRET-basedsystem,aswellasreagentswithincreasedwatersolubility37.

While still the reaction of choice for awide range of bio-conjugation applications, the slowkineticsoftheStaudingerligation,togetherwiththeoxidationliabilityofitsphosphinecompounds,remains an unsolved problem and an obstacle for in vivo chemistry. Consequently,most researchgroupsturnedtheirattentiontotheothermodeofbio-orthogonalreactivityexhibitedbytheazide:its1,3-dipolarcycloadditionwithalkynes.


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1.3.2. Copper-catalyzedazido-alkynecycloaddition(CuAAC)The1,3-dipolar [3+2]cycloadditionofazidesandalkyneswas first reported in189338.But it

wasonly70yearslaterthatthekineticsandmechanismofthereactionwasdescribedbyHuisgen39,whohadhisnamedbestoweduponthisazido-alkynecycloaddition(AAC).

In2001,Sharplessandhiscollaboratorssetouttogeneratesubstancesbyjoiningsmallunitstogetherwith heteroatom links (C-X-C)with the purpose to develop an expanding set of selectiveand modular “blocks” that would reliably work in both small and large-scale applications. Theytermedthisapproachas“clickchemistry”anddefinedastringentsetofcriteria forthisprocess30.The reaction should be modular, wide-scoped, generate high yields with little or inoffensivebyproducts40,stableunderphysiologicalbufferconditionsandinsensitivetooxygen.Therefore,clickreactionsachievetheir requiredcharacteristicsbyhavingahighthermodynamicdriving force,as ifbeing“spring-loaded”forasingletargetinasingletrajectory.Amongthesewerethecycloadditionsofunsaturatedspecies,especiallythe1,3-dipolarcycloadditionreaction40.

Beingsmall,incapableofsignificanthydrogenbondingandrelativelynon-polar,bothazideandalkyneareunlikely to significantly change thepropertiesof structures towhich theyareattached.Additionally,azidesandalkynesarestableinthepresenceofsurroundingnucleophiles,electrophilesandsolventscommontostandardreactionconditionsandcomplexbiologicalmedia.However, therate of this reaction is quite slow, requiring prolonged heating for inactivated alkynes. The AACprocesswasacceleratedbycatalysiswithcopper(I)orruthenium(II)complexes.Becauseofitshighrate and use of the small and easily synthesized azide and terminal alkyne groups, the copper-catalyzed(CuAAC)versionhasbeenthemostwidelyused40.

ThereactionmechanismwasinitiallyproposedbyHimoandSharpless41andrecentlyrefinedby Worrell and Fokin 42. Copper catalysts change the mechanism and outcome of the thermalreaction of terminal or internal alkynes with organic azides, converting it to a sequence of stepswhichculminatesintheformationofadi-copperintermediate(Figure1-9).

Figure1-9:Mechanismofthecopper-catalyzedazido-alkynecycloaddition(CuAAC)


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TherateofCuAACisincreasedbyafactorof107relativetothenon-catalyzedprocess,makingitconvenientlyfastandbelowroomtemperature43.

By 2010, copper stood out as the only metal for the reliable, facile and 1,4-regiospecficcatalysis of the azide-alkyne cycloaddition. Fokin’s group at the Scripps Institute researchedcomplexes of all first-row transition elements and complexes of Ag(I), Pd(0/II), Pt(II), Au(I/III), andHg(II),amongothers,haveallfailedtoproducetriazolesinsyntheticallyusefulyields;theireffectontherateandselectivityofthecycloadditionwasatbestmarginallynoticeable43.

In2005, rutheniumcyclopentadienylcomplexeswere foundtocatalyzethe formationof thecomplementary 1,5-disubstituted triazole from azides and terminal alkynes, and also to engageinternal alkynes in the cycloaddition 44. While the scope and functional group compatibility ofruthenium azido-alkyne cycloaddition (RuAAC) are excellent, the reaction ismore sensitive to thesolventsandthestericdemandsoftheazidesubstituentsthanCuAAC43.ApplicationsofRuAACarestillscarce44.

Meldal and Tornøe 45 published an extensive review on CuAAC showing the effects of thecoppersourceinthereactionandpotentialsidereactionsthatmaypreventthisapproachtobeusedin living cells, which ultimately lead to the introduction of copper ligands to perform thecycloadditionsafelyinintactcells.

1.3.2.1. Coppercatalystsandligands

A wide range of experimental conditions for the CuAAC have been employed since itsdiscovery, underscoring the robustness of the process and its compatibility with most functionalgroups,solvents,andadditivesregardlessofthesourceofthecatalyst.Thechoiceofthecatalystisdictated by the particular requirements of the experiment, and usually many combinations willproduce desired results. Copper(I) salts (iodide, bromide, chloride, acetate) and coordinationcomplexeshavebeencommonlyemployed43.

Copper(II) salts and coordination complexes are not competent catalysts. Cupric salts andcoordinationcomplexesarewell-knownoxidizingagents fororganic compounds.Alcohols,amines,aldehydes,thiols,phenols,andcarboxylicacidsmayreadilybeoxidizedbythecupricion,reducingitto the catalytically active copper(I) species in the process 43. Still, performing the reaction in thepresenceofmild oxidants allows capture of the triazolyl copper intermediates. Ascorbate, amildreductant,wasintroducedbyRostovtsevetal.46asaconvenientandpracticalalternativetooxygen-free conditions. Its combination with a copper(II) salt, such as the readily available and stablecopper(II)sulfatepentahydrate(CuSO4�5H2O)orcopper(II)acetate(Cu(CH3COO)2),hasbeenquicklyacceptedasthemethodofchoiceforpreparativesynthesisof1,2,3-triazoles.Waterappearstobeanideal solvent capable of supporting copper(I) acetylides (Cu2C2) in their reactive state, especiallywhentheyareformedinsitu.The‘‘aqueousascorbate’’procedureoftenfurnishestriazoleproductsinnearlyquantitativeyieldandgreaterthan90%purity,withouttheneedforligandsoradditivesorprotection of the reactionmixture from oxygen 43. Of course, copper(I) salts can also be used incombination with ascorbate, wherein it converts any oxidized copper(II) species back to thecatalyticallyactive+1oxidationstate.


25

Manyothercoppercomplexesinvolvingligandshavebeenreportedascatalystsormediatorsof the CuAAC reaction.Many reported reactions are performed underwidely differing conditions,makingquantitativecomparisonsofligandsperformancedifficult.ThelargestandmostsuccessfulclassofligandsforCuAACcatalysis istheone containing heterocyclic donors. The need for these ligandswasparticularly evident for reactions involving biological molecules thatare handled in water in low concentrations and are not stable toheating.

Despite the experimental simplicity and efficiency of the‘‘ascorbate’’procedure,therateoftheCuAACreactionintheabsenceof an accelerating ligand is simply not high enough whenconcentrationsofreactantsare low.Thefirstgeneral solutiontothebio-conjugation problem was provided by tris(benzyltriazolyl)methylamine ligand (TBTA, CC 16 on page 125), prepared using the CuAACreactionandintroducedshortlyafteritsdiscovery47.Afteritsutilityinbio-conjugationwasdemonstratedby theefficient attachmentof60alkyne-containing fluorescent dye molecules to the azide-labeledcowpea mosaic virus 48 it was widely adopted for use with suchbiological entities as nucleic acids, proteins, E. coli bacteria, andmammaliancells43.

ThepoorsolubilityofTBTAinwaterpromptedthedevelopmentofmorepolaranalogues suchas tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, CC 17 on page 125) 49 and a bis(tert-butyltriazoly)ethanolligand(BTTE,CC18onpage125)50thatshowedtremendousimprovement in reaction kinetics compared to the ligand-freecycloadditioninbiologicalconditions.

Despitetheacceleratedkineticsandreducedcoppertoxicitybytheintroductionof ligandsintheCuAACreaction,theBertozzigroupdevelopedmorebio-friendly copper-free cycloadditionsbyusing theintrinsicenergyofstrainedcyclicalkynestodrivethereaction51.

1.3.3. Metal-freeclickcycloadditionsExogenousmetalscanhavemildtoseverecytotoxiceffectsandcanthusdisturbthedelicate

metabolicbalanceof the systemsbeing studied.Copper, forexample,exerts its toxicitymainlyviafreeradical-inducedoxidativedamage52.

Basedonknowledgethatcyclooctyneandphenylazide“proceededlikeanexplosiontogiveasingle product” – the triazole, Bertozzi’s lab explored this new click chemistry field to circumventmetaltoxicityoftheclassicalcycloadditions.

Figure 1-10: CuAAC-acceleratingligandsofchoice.

TBTA

THPTA

BTTE


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1.3.3.1. Strain-promotedazido-alkynecycloaddition(SpAAC)

The first cyclooctyne that Bertozzi evaluated underwent cycloadditionwith azides to give atriazole, and although its reaction kineticswas vastly accelerated compared to the linear alkynes,therewasroomforimprovement51.Theysoughttoreducetheelectrondensityofthealkynebondbyintroducingafluorineatomtotheringsystem,andthisincreasedthereactionrateconstantby3-fold.Withtheadditionofasecondfluorineatomtothering,tocreatethecompoundnamedDIFO(Figure1-11),theyobservedamarkedincreaseinrateconstant,makingthereaction60timesfasterthantheparentcompound28.WhiletherateofreactionforDIFOwasexceptional,thecompound’ssolubility inwaterwas less than ideal. They then started to fine-tune themoleculeswithdifferentheteroatomswithin the ring structure to achieve a good solubility/reactivity balance, and found agood candidate with DIMAC 53. At the same time other groups increased the reactivity of thecyclooctynesvia introductionofneighboringbenzyl rings 54.Researchadvancedrapidlyanda largemyriad of strained alkynes surfaced where in most cases the increased reactivity meant lesshydrophilicityandvice-versa28,55,56.

Figure1-11:Strain-promotedazido-alkynecycloaddition.ExamplereactionbetweenDIFOandanazide.

Therearenumerousapplicationsofcyclooctynesinthecontextofbiologicalsystems.Bertozzi

et al. proved its applicability on modification of purified proteins 51, later on the method wasextended to in vivo experiments 55. Furthermore, SpAACwas used to image tumors in livingmicewiththehelpofnanoparticles57and18Fpositronemissiontomography(PET)58.

Despite some problems in the earlier development of SpAAC such as slow kinetics andspontaneousdefluorinationofreagentswhichwereovercomebyreagentoptimization,thismethodhasprovenitssuitabilityforinvivolabelingseveraltimes.TheissueofincreasedunspecificlabelingcomparedtoStaudingerligationremainstobesolved.

1.3.3.2. Tetrazineligation

While others were working with alkynes to form triazoles upon cycloaddition with azides,Rutjesandco-workers59developedanalkynesurrogatethatformsa1,2,3-triazoleproductinatwo-stepreactionthatincludedaretro-Diels-Alderreaction*.

*TheDiels-Alderreactionisa[4+2]cycloadditionofaconjugateddieneandadienophile(analkeneoralkyne).E.g.:


27

In 2008, the Fox 60 and Hildebrand 61 groups put the inverse electron demand Diels-Alderreaction to use in bio-orthogonal ligation chemistry using a tetrazine to react with a trans-cycloocteneandnorbornene,respectively(Figure1-12).

Figure1-12:Tetrazineligation.Example reactions between tetrazines and a trans-cyclooctene (A) as published in Fox et al. 60 and a norbornene (B) as published byHildebrandandco-workers61.

Electron-richtetrazinesarelesssusceptibletohydrolysisandattackbybiologicalnucleophiles,

andare thusmoredesirable foruse invivoand intracellularenvironments, inparticular.Stabilizedtetrazines have recently been introduced into recombinant proteins via unnatural amino acidmutagenesis 62. The heterocyclic amino acid derivatives integrated biologicalmolecules that werethendetectedwithstrainedalkenes63.Ahead-to-headcomparisonofthereactionsofthesevariousalkenes and tetrazines has not been reported, but combining the best feature of each approachcouldaddvaluabletoolstothefield28.

The tetrazine click is showingpromising results in thebio-orthogonal chemical ligation field,particularly in the detection of fast biological processes. However, the bulkiness of themoleculesinvolvedcouldhindersomeapplicationsofthetechnique.

Veryrecentlytheuseofclickreactionsforchemicallytriggeredreleaseofdrugs invivobasedontetrazinesandtrans-cyclooctenesisbeingstudied64,65.

Manyotherchemical ligationshavebeenexplored tobeused inbiological settings, suchas

cycloadditionsinvolvingnitrileiminesornitrileylides;azido-alkynecycloadditionsusingothermetalsas catalyst; thiol-ene coupling;palladium-catalyzed coupling reactions; andphoto-inducibledipolarcycloadditions. In fact, a cooperation partner of the BioChemLig network in which the researchpresented in this dissertation was included, published recently novel chemical reactions withpotentialtooriginatenewbio-orthogonalligations66.

The ligationspresented in thepreviousparagraphs represent themostusedand relevant inbio-orthogonalclickchemistryresearch.

Chapter2:Labelingaproteinusingclickchemistry|Introduction

28

2. Labelingaproteinusingclickchemistry2.1. INTRODUCTION

Cyclic adenosine monophosphate (cAMP) is an important biological second messengermolecule that regulates several cellular events and complex biological processes such asmemoryconsolidation,immunesystemregulation,insulinsecretionandcardiacfrequency67.cAMPwasfirstdescribedin1956byRalletal.whentheyobservedthattheincreasedproductionofphosphorylaseinliverhomogenatesuponhormonalstimulationwasmediatedbyaheat-stablefactor68.Soonafterthatdiscovery, the samegroupcharacterized that factor tobeanadenine ribonucleotide 69,70 thatwasidenticaltoaproductisolatedatthesametimebyCooketal.fromthealkalinedegradationofadenosinetriphosphate(ATP)71.

cAMPisproducedincellsfromATPbyadenylylcyclasesthataremainlylocatedintheplasmamembrane and these can be regulated in different ways such as via G protein-coupled receptors(GPCRs) that respond to external stimuli in the form of hormones or neurotransmitters 72,73;intracellularCa2+andbicarbonateregulatecAMPproductionviaasolubleadenylylcyclase74,75;andpost-translational modifications such as phosphorylation can also affect the activity of adenylylcyclases 76,77. The effector function of cAMP is terminated by specific phosphodiesterases thatcatalyze thehydrolysisofcAMP intoAMP69,controlling thepoolofavailablecAMPandregulatingthe signaling cascade. Additional mechanisms of cAMP production and clearance have beendescribed 72,76, suchas the targetingof specific adenylyl cyclases tomembranemicrodomains thatcontributetotheformationofcAMPcompartmentswithclinicalrelevance78,andtheeffluxofcAMPtotheextracellularspacethroughamultidrugresistancetransportsystem79.

ProteinkinaseA(PKA)wasthefirstdown-streamtargetforcAMPtobeidentifiedbyWalshetal.in196880.PKAisaheterotetramercomposedoftworegulatoryandtwocatalyticsubunitswhosemultiplesubunits(RIα,RIβ,RIIα,RIIβandCα,Cβ,Cγ)possessdistinctphysicalandbiologicalproperties,are differentially expressed, and are able to form different isoforms of PKA holoenzymes 81.Activation of PKA involves the cooperative binding of four cAMPmolecules to the two regulatorysubunits,inducingaconformationalchangeandreleasingbothcatalyticsubunitsthatarethenabletophosphorylatetheirproteinsubstrates82.TheregulatorysubunitsofPKAarealsoresponsibleforthe binding of scaffolding proteins calledA-kinase anchoring proteins (AKAPs) that localize PKA tospecific sites in close proximity to its protein substrates 82,83. A scheme of cAMP synthesis andsignalingviaPKAisillustratedinFigure2-1,onpage29.

AdditionaltargetsforcAMPhavebeendescribedthataffecttheelectricalactivityofcells,suchashyperpolarization-activatedcyclicnucleotide-gated (HCN) channels 84 anddirectlymodulatedbycyclic nucleotides (CNG) channels 85.More recently, exchangeproteins activatedby cAMP (EPACs)thatmodulatetheactivityofthesmallGTPasesRap1andRap2,havebeenestablishedasafamilyofproteinsthatdirectlybindcAMP86,87.


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Figure2-1:cAMPproductionandactivationofPKA.LigandbindingtoGprotein-coupledreceptors(GPRCRs)activatesneighboringadenylylcyclasesandgeneratespoolsofcAMPfromATP.ThedistributionandconcentrationgradientofcAMPislimitedbyphosphodiesterases.BindingofcAMPtotheregulatory(R)subunitsofproteinkinaseA(PKA)releasesthecatalytic(C)subunitsoftheheterotetramerallowingthemtophosphorylatetargetsubstrates.CertainadenylylcyclasesandGPCRsareconfinedtomembranemicrodomainsinassociationwithintracellularorganellesorcytoskeletalconstituents.ThesubcellularstructuresharborspecificisoformsofPKAthatarelocalizedinthevicinityofthereceptorandcyclasethroughanchoringviaAkinaseanchoringproteins(AKAPs).Thesemechanismsservetolocalizeandlimittheassemblyofthepathwaytoadefinedareaofthecellclosetothesubstrate.AdaptedfromTaskénetal.81usingServierMedicalArttemplates.

Massspectrometry-basedstudiesoncAMP-bindingproteinshaveidentifiedfurthertargetsfor

thesmallmolecule,inadditiontothepreviouslydescribed.Pull-downstudiesperformedonHEK293cellsanddifferentrattissuelysates88–90usedcAMPimmobilizedonagarosebeadstoenrich,isolateand identify the directly interacting proteins and their interaction partners. The results show thatdifferent isoforms of the regulatory subunit of PKA are enriched as well as a set of AKAPs andphosphodiesterases.Oneparticularenrichedprotein,theoncoproteinsphingosinekinaseinteractingprotein (SKIP) that was not known at the time to be involved in the cAMP pathway, was laterestablished as a new AKAP 91. The authors also concluded that the cyclic nucleotide proteome iscontaminatedbyabundantproteins88andsotheysequentiallyelutedthepulled-downproteinswithdifferent nucleotides to further enrich the true cAMP binders. Pull-down strategies with bead-immobilizedsmall-moleculesarepronetopresenthighunspecificbackgroundbecausethewashingconditions have to be mild and usually consist of only several washes with lysis buffer. Thesestrategies are also little effective in the enrichment of membrane proteins with multiple trans-membranesegments.


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In the CCMS approach, as described earlier (1.2.4 Capture compound mass spectrometry(CCMS),onpage18),washingconditionscanbemorestringent(highsaltanddetergent)becauseofthecovalentcrosslinkbetweenthemodifiedsmallmoleculeandthetarget,increasingthesignal-to-noiseratioofthetruebindersofthesmallmolecule.Havingaparallelcompetitionassay,inadditiontothestringentwashing,allowsalsoforamoreaccuratedeterminationofrealtargets.

ThecAMP-interactingproteomewas studiedby classicalCCMS inHepG2cells lysateand ratbrainsynaptosomalfractions12.Aspreviousstudieshaveshown,isoformsoftheregulatorysubunitsofPKAwere identifiedusingCCMSalongwithseveralAKAPs,PKGandEPACs.Thenoveltywasthesuccess in identifying endogenously expressing HCN channels from the synaptosomal fractions,whichwas a knownbinding partner of cAMPbut not demonstrated before bymass-spectrometryproteomics. Some of the enriched proteins in these experiments were also competed with othernucleotides, confirming the promiscuous binding observed before and theymay aswell be directbindersofcAMPbut lack thecyclicnucleotidebindingdomainpresent in theclassic targetsof thesmallmolecule.

With the knowledge of the strong interaction between cAMP and PKA via its regulatorysubunits, a purified recombinant human PKARIwas acquired and a cAMP analogwas synthesizedbearingaphoto-reactivemoietyandaterminalalkynereactivityfunction(CC2,onpage122)fortheestablishmentofanewCCMSworkflowusingatwo-stepbio-orthogonalchemicalligationmethod.

Chapter2:Labelingaproteinusingclickchemistry|Results

31

2.2. RESULTS2.2.1. Establishingandoptimizingclick-captureofPKARIusingacAMPCC

As previously described in the General Introduction (1.3 Click chemistry and bio-orthogonalligations,onpage21),thereareanumberofbio-orthogonalligationsdescribedintheliterature,buttodatethemostbroadlyutilizedhasbeenthecopperassistedazido-alkynecycloaddition(CuAAC).This “click” reaction was chosen to establish a labeling method of a protein using a capturecompoundcontainingtheselectivityandreactivityfunctionsbutwithanalkyneterminalfunctioninplaceofthesortingfunctionasinaclassicalcapturecompound.

To achieve a successful labeling, different parameters such as capture compoundconcentration, time of the click reaction, copper concentration and copper ligands wereindependentlyvaried.

Thecapturecompounds,competitorand ligandsutilized in thischapterare illustrated in theSpecificMethodssection(2.3.1Structuresofusedcompounds,onpage48)

As a starting point to establish this method in a CCMS workflow, a cAMP-alkyne capturecompound(CC2)waschosenandallreactionoptimizationstepswereperformedinarecombinantPKARIsolution.Theprobewasclickedtoanazide-biotin (CC3) forvisualizationandcaptureofthetarget.Asareference,aclassicalcAMP-capturecompound(CC1)wasusedinparallel.

2.2.1.1. EffectofcAMP-alkynecapturecompoundonclickyieldsinaPKARIsolution

Initialclick reactionconditionswerebasedonapublicationbySpeersetal. 23.The firststepwas a concentration-dependent assessment starting from the generic condition to determine theeffectoftheconcentrationofcAMP-alkynecapturecompoundonthelabelingofrecombinantPKARIusingbiotinasalabel.

16pmolofPKARIwasincubatedwithcAMP-alkynecapturecompound(CC2)at5µM,2.5µM,1µM,0.5µMornoneforunspecificlabelingcontrol.Asamplecontaining5µMofcAMPfullclassicalcompound(CC1)wasusedaslabelingreference.After30minequilibration,sampleswereirradiatedfor10minat310nminaCaproBox®.Next,tothecAMP-alkynecontainingsamplesthefollowingwasadded:10µMbiotin-azide(CC3),50µMCuSO4,250µMTBTA(CC16)and2.5mMsodiumascorbate.The click reactionwas performed for 30minwith shaking. To a duplicate sample of cAMP-alkynecapture compound at 5 µM, all click reagents except for the biotin-azidewere added. The resultswerevisualizedbySDS-PAGEfollowedbyBlottingusingastreptavidin-horseradishperoxidase(HRP)solution for chemiluminescence readout detected on x-ray film (hereafter referred as biotin blot).BandintensitywasmeasuredusingImageJsoftware.

ThebiotinblotshowninFigure2-2,onpage32,revealsthattheclickreactiontakesplaceandisdependentontheconcentrationofthecAMP-alkynecapturecompound.Figure2-2suggeststhatthe click reaction yield with these initial parameters is less than 50% when using the equivalentconcentrationof cAMP-alkynecapturecompoundandcomparing it to the labeling intensityof theclassical full capture compound (Figure 2-2, lane 1 vs. lane 4). Decreasing the clickable capturecompound’s concentration decreases the intensity of the biotin signal, suggesting the biotin-azidecounterpart is in excess and does not cross-link to the protein directly (Figure 2-2, lanes 5 to 7).Additionally, it can be observed that no unspecific reaction occurs in the absence of the alkyne


32

capture compound (Figure2-2, lane2), confirming thebio-orthogonal characteristicof theCuAAC,noristhereanunspecificsignalintheabsenceofthebiotinlabel(Figure2-2,lane3).

Figure 2-2: Labeling of recombinant PKARI using CuAAC in a serialdilutionofcAMP-alkynecapturecompound.16 pmol of PKARI was incubated with: lane 1 – 5 µM full cAMPcapturecompound(CC); lane2–nocapturecompound; lane3andlane 4 – 5 µM cAMP-alkyne CC; lane 5 – 2.5 µM cAMP-alkyne CC;lane 6 – 1 µM cAMP-akyne CC; lane 7 – 0.5 µM cAMP-alkyne CC.CuAACreactionwascarriedout in lanes2to7using50µMCuSO4,250µMTBTA,2.5mMsodiumascorbateand10µMbiotin-azide(inlane 3 equivalent volume of water was added instead). Bar plotdisplaysrelative intensityofbandsmeasuredbydensitometryusingfullcapturecompound(lane1)asreference.

This initial experiment seems to suggest

that either the new cAMP-alkyne CC binds withless affinity than the original full CC or that theclickreactioninducesproteindegradationorloss.To control the latter, subsequent experimentswereprobedforPKARIusingaspecificantibody.

Inanewexperiment,PKARIsamplescontainingthedifferentconcentrationsofcAMP-alkyne

capturecompoundwereduplicatedandpre-incubatedwith1mMfreecAMPtoactascompetitor(Figure2-3).Subsequenttreatmentwasperformedsimilarlyacrosssamplesasdescribedpreviously.InFigure2-3 intensitybarswereplottedasanormalizationofthebiotin intensityagainstthetotalPKARIsignal ineachlane.Thefirstobservationisthatproteinamountisvariableacrosstheclickedsamples(Figure2-3,PKARIpanel,lanes3–12).Moreover,consistentcompetitionwasnotobservedin this experiment, except perhaps in samples containing the least amount of cAMP-alkynecompound (Figure2-3, lanes11and12),but thedifferenceof theamountofPKARIbetweenbothsamples does not allow a correct evaluation of different biotin signals which allows for theassessmentofcompetition.

Figure 2-3: Labeling ofrecombinant PKARI usingCuAAC in a serial dilution ofcAMP-alkyne capturecompoundwithcorrespondingcompetitionsamples.16 pmol of PKARI wasincubatedwith (C) or without(A) 1 mM cAMP competitor.Samples in lane 1 and lane 2were labeled with 5 µM fullcAMPcapturecompound(CC);samples in lanes3to12wereincubatedwith serialdilutionsof cAMP-alkyne CC asindicated.CuAACreactionwascarried out in lanes 3 to 12using 250µMCuSO4, 250µMTBTA, 2.5 mM sodiumascorbate and 10 µM biotin-azide. Bar plot displaysintensity of biotin normalizedto intensity of PKARI incorrespondingsample.


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Theexperiments illustrated inFigure2-2andFigure2-3,onpage32,demonstrated that thelabelingsignalobtainedwithafullclassicalcapturecompoundismoreintensethantheoneobtainedwith the new CuAACmethod using the cAMP/PKARI system and the original Speers et al. CuAACconditions23.Furtheroptimizationsontheindividualcomponentsoftheclickreactionarenecessaryandtheresultsfromthoseexperimentsaredescribedahead.

2.2.1.2. EffectofcoppersulfateconcentrationonclickyieldsinaPKARIsolution

TodeterminetheeffectofcoppersulfateconcentrationonthebiotinylationofPKARIusingacAMP alkyne compound a new experiment was designed with the knowledge obtained from theinitialexperiments.

16pmolofPKARIwasincubatedwith5µMcAMP-alkynecapturecompound(CC2).Asamplecontaining5µMofcAMPfullclassicalcompound(CC1)wasusedaslabelingreference.After30minequilibration, samples were irradiated for 10min at 310 nm in a CaproBox®. Next, to the cAMP-alkynecontainingsamplesthefollowingwasadded:10µMbiotin-azide(CC3),250µMTBTA(CC16),2.5mMsodiumascorbateanddifferentCuSO4concentrations(0,50µM,250µM,500µMand750µM).Theclickreactionwasperformedfor30minwithshaking.ToanadditionalsamplecontainingthecAMP-alkynecapturecompound,allclickreagentsexcept forthebiotin-azidewereadded.Theresults were visualized by SDS-PAGE followed by Blotting using a streptavidin-HRP solution forchemiluminescencereadoutonx-rayfilm.BandintensitywasmeasuredusingImageJsoftware.

Figure 2-4: Effect of copper sulfateconcentration on the click labeling ofrecombinant PKARI using a cAMP-alkynecapturecompound.16 pmol of PKARI was incubated with 5µM full cAMP capture compound (CC)(lane1)or5µMofcAMP-alkyneCC(lanes2–7).CuAACreactionwascarriedout inlanes2to7using250µMTBTA,2.5mMsodiumascorbate, 10µMbiotin-azide (inlane 2 equivalent volume of water wasadded instead) and increasingconcentrationsofCuSO4(lanes3–7).Barplot displays relative intensity of bandsmeasured by densitometry using fullcapturecompound(lane1)asreference.

As depicted in Figure 2-4, in the conditions tested, the increasing concentration of copper

sulfateintheclickreactiononlyyieldsthehighestsignalat250µMofCuSO4.Evenatthismaximum,the signal intensityonlyaccounts for≈10%of the labelingobtainedwith theclassical full capturecompound(Figure2-4,lane5).

TodetermineifthelowyieldreactionwasduetothepresenceofthecopperligandTBTAoritsratiotocoppersulfate,newsampleswerepreparedandanalyzedaspreviouslydescribed.


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Figure2-5:EffectofcopperligandTBTAontheclicklabelingofrecombinantPKARIusingacAMP-alkynecapturecompound.16pmolofPKARIwas incubatedwith (C)orwithout (A)1mMcAMPcompetitor.Sampleswere incubatedwith5µMfull cAMPcapturecompound(CC)(lanes1and2),5µMofcAMP-alkyneCC(lanes3to8andlanes11and12),ornone(lanes9and10).CuAACreactionwascarriedoutinlanes3to12using2.5mMsodiumascorbate,10µMbiotin-azide(exceptlanes7-8),250µMTBTA(exceptlanes5and6)andeither250µMCuSO4(lanes3to10)or50µMCuSO4(lanes11and12).Barplotdisplaysintensityofbiotin(panelA),PKARI(panelB)andnormalizedbiotinintensitytoPKARIincorrespondingsample(panelC).

In the biotin blot illustrated in Figure 2-5, we can observe that labeling is reduced in thecompetition sample of the classical cAMP capture compound (Figure 2-5, panel A, lane 2) whencompared to the corresponding assay (Figure 2-5, panel A, lane 1) as is expected in a CCMSexperiment. The samples containing copper sulfate and TBTA at 1:1 ratio present a similarcompetitionpattern,althoughtheoverallintensitiesofthebiotinsignalarelower(Figure2-5,panelA, lanes 3 and 4). With higher labeling intensities, although no visible competition, we have thesamples reacted with CuSO4:TBTA at 1:5 (Figure 2-5, panel A, lanes 11 and 12) and even higher,comparabletothefullclassicalcapturecompound,thesampleswithoutTBTA(Figure2-5,panelA,lanes5and6).

The signal for total PKARI is variable across the samples (Figure 2-5, panel B), as seenpreviouslyinFigure2-3,onpage32.Duetothisvariability,thesignalobtainedinthebiotinblotwasnormalized to the intensity of PKARI detected in the corresponding sample. The results of thisnormalizationareplotted inFigure2-5,panelC.Theoutcomeof thenormalizationshowsthat theclickedsampleswithoutTBTApresentthehighestbiotinylationyieldsbutnocompetitionisobserved(Figure2-5,panelC,lanes5and6).Thiscouldbeaskewedresultfromthemuchlowerabundanceofthe signal from the total protein in these samples (Figure 2-5, panel B, lanes 5 and 6). Theseobservationssuggestthatfreecoppersulfate,intheabsenceofaligand,promotesthecycloadditionreactionbutatacostofdegradingprotein.ThepresenceofTBTAminimizestheproteindegradationbutalsodiminishestheyieldoftheCuAACreaction.

Overall,theseresultsseemtoindicatethatTBTAisnotanadequatecopperligandforourclickapproachworkflow. New ligands should be investigated if they produce amore efficient reactionyieldintheCuAAC.


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2.2.1.3. TestingtimeofCuAACreactiononclickyieldsandproteinstabilityinaPKARIsolution

The yields of the click reaction in a semi-complex system such as the cAMP-alkyne capturecompoundandbiotin-azideinarecombinantPKARIsolutioncouldbetime-dependent,butaproteinin solutionwill tend to degrade throughout this process. It is therefore important to determine atime-point for theclickreactionwherethe labelingyieldsaresatisfactoryandthetargetprotein ismostlyintactinthereactionvial.

Previousresultsdescribedinthepreviousparagraph2.2.1.2andillustratedinFigure2-5,havesuggestedthatthecopper ligandsofarutilizedwasnotadequateforthenewclickworkflowtobeimplemented.Hongetal.producedawater-solublecopperligand,THPTA(CC17),andshowedthatithashigherkineticsintheCuAACreactionwhencomparedtothetraditionallyutilizedTBTA(CC16)49.WesetouttodeterminetheoptimaltimefortheclickreactionofacAMP-alkynecapturecompoundtoabiotin-azideinarecombinantPKARIsolutionusingthenewligandTHPTA.

RecombinantPKARIwasincubatedwithcAMP-alkynecapturecompound(CC2)at5µM,cAMPfull classical compound (CC 1) at 5 µM or none for unspecific labeling control. After 30 minequilibration, samples were irradiated for 10min at 310 nm in a CaproBox®. Next, to the cAMP-alkynecontainingsamplesthefollowingwasadded:10µMbiotin-azide(CC3),250µMCuSO4,250µMTHPTA(CC17)and2.5mMsodiumascorbate.ToanadditionalsamplecontainingcAMP-alkynecapture compound at 5 µM, all click reagents except for the biotin-azide were added. The clickreaction was performed up until 180 min shaking and aliquots were collect at determined timepoints. The results were visualized by SDS-PAGE followed by Blotting using a streptavidin-HRPsolution for chemiluminescence readout on x-ray film. Band intensitywasmeasured using ImageJsoftware.

Figure2-6:LabelingofrecombinantPKARIusingacAMP-alkynecapturecompoundatdifferentCuAACreactiontimes.PKARIwasincubatedwith5µMfullcAMPcapturecompound(CC)(lanes1and2),5µMofcAMP-alkyneCC(lanes5–14)ornone(lanes3and4).AliquotswererecoveredatdescribedtimepointsoftheCuAACreactioncontaining250µMCuSO4,250µMTHPTA,10µMbiotin-azide(inlanes5and6equivalentvolumeofwaterwasaddedinstead)and2.5mMsodiumascorbate.SampleswiththeclassicalfullcAMPCCwere treated similarly andalso collectedatdifferent timepoints. Barplot displays intensityof biotin (panelA), PKARI (panelB) andnormalizedbiotinintensitytoPKARIincorrespondingsample(panelC).


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Figure2-6,onpage35,showsthatdetectionofPKARIdecreasesasthetimeofCuAACreactionincreases(Figure2-6,panelB),mostlikelyduetoproteindegradation.Wecanalsoobserveintheseresults that the degradation is independent of the presence of either the cAMP-alkyne CC or thebiotin-azidecounterpart(Figure2-6,panelB,lanes3–6).InpanelAofFigure2-6,wecanseethatthebiotinlabelingobtainedviatheCuAACreactionreachesamaximumat60min(Figure2-6,panelA,lane11),althoughthissignalisstillafractionoftheoneobtainedbyusingtheclassicalfullcapturecompound(Figure2-6,panelA,lanes1and2).WhenthebiotinsignalisnormalizedtotheamountofPKARIdetected in thesample (Figure2-6,panelC)wecanobserveanapproximationof thesignalintensitybetweentheclassicalfullcapturecompoundandtheclickablecompoundat,e.g.,60minofreaction which was previously mentioned as the maximum biotin signal in the CuAAC reactionsamplesmeasured (Figure2-6,panelC, lane11).Highernormalized intensities canbeobservedat120minof reaction (Figure2-6, panelC, lane13), but these results areoverestimateddue to thedecreasingintensityofthetotalPKARIdetectionwhencomparedtotheinitialtimepoint(Figure2-6,panelB,lanes7and13),probablyduetoproteindegradation,precipitationordenaturation.

ThechosenCuAACreactiontimeforfutureexperimentswasbetween30and60min.

2.2.1.4. Optimizationofbiotin-azideconcentrationinCuAACreactionfordetectionofPKARI

Inordertoachievemaximumreactionyield,weresearchedtheinfluenceoftheconcentrationof biotin-azide in the CuAAC reaction using a recombinant PKARI solution and the cAMP-alkynecapturecompoundastheprimaryprobe.

PKARIsamplesforcompetitionassayswerepre-incubatedwith1mMfreecAMP(CC13)andtonormalassaysamplestheequivalentvolumeofwaterwasadded.Competitionandnormalassaysamples were then incubatedwith with 5 µM cAMP-alkyne capture compound (CC 2). Assay andcompetitionsamplescontaining5µMofcAMPfullclassicalcompound(CC1)wereusedaslabelingreference.After30minequilibration,sampleswereirradiatedfor10minat310nminaCaproBox®.Next,tothecAMP-alkynecontainingsamplesthefollowingwasadded:250µMcoppersulfate,250µMTHPTA(CC17),2.5mMsodiumascorbateanddifferentbiotin-azideconcentrations(0,5µM,10µM, 20 µM and 30 µM). The click reaction was performed for 30 min shaking. To an additionalcontrol sample containing the cAMP-alkyne capture compound, all click reagents except for thebiotin-azide were added. The results were visualized by SDS-PAGE followed by Blotting using astreptavidin-HRP solution for chemiluminescence readout on x-ray film. Band intensity wasquantifiedusingtheImageJsoftware.


37

Figure2-7:LabelingofrecombinantPKARIusingCuAACwithdifferentbiotin-azideconcentrations.16pmolofPKARIwasincubatedwith(C)orwithout(A)1mMcAMPcompetitor.Samplesinlane1andlane2werelabeledwith5µMfullcAMPcapturecompound(CC);samplesinlanes5to14wereincubatedwith5µMcAMP-alkyneCCandsamplesinlanes3and4wereusedascontrol.CuAACreactionwascarriedout in lanes3to14using250µMCuSO4,250µMTHPTA,2.5mMsodiumascorbateandvaryingconcentrationsofbiotin-azideasindicated.Barplotdisplaysintensityofbiotin(panelA),PKARI(panelB)andnormalizedbiotinintensitytoPKARIincorrespondingsample(panelC).

Figure2-7showsthebio-orthogonalcharacteristicoftheCuAACreactiondemonstratedbythe

absenceofabiotinylationsignalinsampleslackingeitherthecAMP-alkynecapturecompoundorthebiotinazide(Figure2-7,panelA,lanes3to6).Thesameblotalsoshowsthebiotinsignalincreasingwithmorebiotin-azide in solutionand saturatingat20µMofazide,which is still a fractionof thesignalobtainedwiththeclassicalfullcapturecompoundbuthigherthaninanypreviouslypresentedresult.Figure2-7alsoshowsthatincreasedamountsofbiotin-azideinsolutionhasaneffectonthedetection of PKARI but the intensity of these samples is always higher than 50% of the signaldetected in the samplewithoutbiotin-azide (Figure2-7,panelB, lanes5and6),except in lane11where detection is considerably lower andwas considered anoutlier. Analyzing the plot obtainedwhenthebiotin labeling isnormalizedtothetotalPKARI(Figure2-7,panelC)wecanobservethatthepre-incubationwithexcessfreecAMPcompetitordidnotproducetheexpectedreductioninthebiotin signal for those sampleswhencompared to theassaypair (e.g., Figure2-7,panelsAandC,lanes1and2).Also,reactingmorethan10µMofbiotin-azideapparentlydoesnotimprovefurtherthebiotinsignalreadout.


38

2.2.1.5. CaptureofPKARIwithaclickablecAMPcapturecompoundusingCuAAC

AftertheconditionsforthebiotinlabelingofPKARIusingaCuAACreactionwereoptimized,asdescribed in the previous paragraphs of this chapter, we set out to capture it using magneticstreptavidinbeads.

RecombinantPKARIwasinitiallyincubatedwith(C)orwithout(A)excessfreecAMP(CC13)for10minandthenaddedtothesamplescAMP-alkynecapturecompound(CC2)at5µMorcAMPfullclassicalcompound(CC1)at5µM.After90minequilibration,sampleswereirradiatedfor10minat310nminaCaproBox®.Next,tothecAMP-alkynecontainingsamplesthefollowingwasadded:10µMbiotin-azide(CC3),50µMCuSO4,250µMTHPTA(CC17)and2.5mMsodiumascorbate.Tothefullclassicalcompoundsamples,thesamevolumeofwaterwasadded.Theclickreactionwasrunfor30min.Analiquotwasremovedandlabeledasinputforvisualizationongel.Totheremainderofthereaction,streptavidinmagneticbeadswereaddedandmixedfor1hour.Intheend,thebeadswerecollectedwith a CaproMag®,washed and boiled in Laemmli buffer to extract the bound proteins.Several fractionsof thesampleswere recovered foranalysis: (1) thesampleaftercross-linking thecapturecompoundbeforebeadswereadded,(2)thebeadswiththecapturedcompoundand(3)theremainder supernatant after beads were recovered. All fractions were visualized by SDS-PAGEfollowedbyBlottingusingastreptavidin-HRPsolutionforchemiluminescencereadoutonx-rayfilm.BandintensitywasmeasuredusingImageJsoftware.

Figure2-8:Click-captureofPKARIusingacAMPalkynecapturecompound.PKARI was pre-incubated with (C) or without (A) excess free cAMP. The samples were then incubated with 5 µM full cAMP capturecompound(CC)(lanes3–8),5µMofcAMP-alkyneCC(lanes9–14)ornone(lanes1and2).Fractionsanalyzed:beforeadditionofbeads(input),beadcapture(beads)andremainingsampleafterbead incubation(supernatant).TheeffectofUV irradiationtoPKARIdetectionwasanalyzed(PKARI@310nm).Barplotdisplaysintensityofbiotin(panelA)andPKARI(panelB).


39

Inthisinitialclick-captureexperimentwealsoanalyzedtheeffectoftheUVradiationusedtocross-link the capture compounds to the targetproteinon thedetectionof PKARI. In fact, theUVradiation decreases the detection of PKARI by 10% (Figure 2-8, panel B, lanes 1 and 2), not byinducingdegradationoftheproteinbutbypromotingdimerizationofPKARI,whichwasobservableasabandnextto130kDamarkeronlyintheirradiatedsample.

When looking at the results obtained using the classical full capture compound (Figure 2-8,lanes3 to8) it is clear thatpre-incubationwithexcesscAMPcompetitorprevents thebiotinylatedcapturecompoundtobindtoPKARIandhencereducethedetectionofbiotininthatsample(Figure2-8, panel A, lane 4) when compared to the normal assay (Figure 2-8, panel A, lane 3). ThiscompetitioneffectisonceagainobservedinthecapturedbeadsfractionandsuccessisconfirmedbythedifferentialdetectionoftotalPKARIbetweenassayandcompetition(Figure2-8,panelB,lanes5and 6). Some PKARI remains in solution after the bead capture with a reversed intensity profile(Figure2-8,panelB, lanes7and8),mainlyduetothefactthat itwasnotcarryingthebiotinylatedcapturecompound(Figure2-8,panelA,lanes7and8)andthereforedidnotbindtothestreptavidinmagneticbeads.

Results fromthenewclick captureworkflowarealso shown (Figure2-8, lanes9 to14).Thebiotinsignalintheassaysampleandalmostundetectableinthecompetitionsamplebeforeaddingthebeads(Figure2-8,panelA,lanes9and10)suggeststhathavingatwostepapproachprovidesfora more evident competition effect. The corresponding PKARI blot for these samples (Figure 2-8,panel B, lanes 9 and 10) shows that the amount of protein present is similar between them andtherefore a true competition effect on the labeling using the new click workflow was obtained.Analyzing thecapturedbeads fractionof theclick samplesnobiotinylation isdetected (Figure2-8,panelA,lanes11and12),whichwouldsuggestthatthetargetproteinwasnotsuccessfullycaptured,but looking at thePKARIblot for the same samples (Figure2-8, panelB, lanes11 and12)we canobservethattheproteinwasindeedsuccessfullycapturedintheassaysampleandisnotdetectedinthecompetition.Thisdiscrepantobservationwouldsuggestthatdetectionofbiotin isnotsensitiveenoughtodeterminewithcertainty thesuccessofacaptureexperimentandshouldbe taken intoaccountinfutureexperiments.Itisalsoimportanttoconsiderthatnon-cross-linkedcompoundsarestill able to pull-down the target and after the sample treatmentnobiotin signal is detected. Theoverall capture yield in thenewclick chemistryworkflow is less thanwhenusing theexhaustivelyoptimizedclassicalcapturebutitappearstobe“cleaner”.

Overall, thenewCuAACcapturemethodofPKARIusingaclickablecAMPcapturecompoundpresented in this experimentwas successful and thisworkflowcanbeusedas a startingpoint forcapturing in more complex systems such as a cellular lysate or living cells. Before increasing thecomplexityofthesystem,anewcopperligandwasintroducedintheworkflowtoincreasetheyieldsoftheCuAACreaction.


40

2.2.1.6. UsingBTTEascopperligandandadjustingreactionpHincreasesPKARIclicklabelingyield

Soriano del Amoet al. demonstrated BTTE (CC 18), a new copper ligand, to have increasedreaction kinetics in comparison to the previously used TBTA (CC 16) and THPTA (CC 17) 50. Weinvestigated the effect of this new ligand on the efficiency of click labeling PKARIwith the cAMP-alkynewiththepreviouslyestablishedparameters.

Upuntilthisexperiment,labelingefficiencyoftheclickmethodwasdirectlycomparedtothenormal classical compound and themaximum labeling achievedwith the cAMP-alkyne compoundwasaround50%oftheoneobtainedwiththeoriginal fullcompound. Inordertodiscardpotentialaffinity differences towards PKARI between the classical and the clickable cAMP compounds, thecAMP-alkyne compound was pre-clicked to the biotin-azide (CC 4) before incubation with therecombinantproteinandusedasanadditionalcontrol.

As seen in Figure 2-9, the pre-clicked

cAMP compound labels PKARI similarly to thefull classical capture compound (Figure 2-9,panel C, lanes 1-2 and5-6) confirming that theaffinity of these molecules to the recombinantproteinisguidedbytheselectivityfunctionandthattherestofthecompoundhasnoimpactonthelabeling.However,whentheCuAACreactionis performed after the cAMP-alkyne is cross-

linked to the PKARI protein there is a substantial decrease in biotin labeling (Figure 2-9, panel C,lanes3and4).Theseobservationssuggestthatthecompoundsremainintactaftertheclickreactionbutwithinaproteinsolutiontheyieldsaredrasticallyreduced.

2.2.1.7. IntroducingTAMRAasadetectionfunctionusingaclickablecAMPcapturecompoundforPKARIlabeling

Biotinhasbeenusedasa labelwithintheprobesusedsofarmainlyduetothefactthattheoriginalcAMPcapturecompound(CC1)carriesitanditwasusedasreferencefortheoptimizationofPKARI labeling using the CuAAC reaction. This molecule is widely used for pull-down/captureexperiments due to its high-affinity towards streptavidin.Workingwith a purified protein such asPKARI presents little challenges when using biotinylation as a readout system, but when movingtowardslabelingproteinswithincomplexmedia,suchascellularlysatesandwholelivingcells,itcanbe a hindrance because these proteomes contain endogenously biotinylated proteins which willinterferewiththereadout.

Figure 2-9: Labeling of PKARI with a cAMP-alkyne capturecompound clicked to a biotin-azide using CuAAC with BTTE ascopperligand.PKARI was pre-incubated with (C) or without (A) excess freecAMP. The samples were then incubated with 5 µM full cAMPcapturecompound(CC)(lanes1and2),5µMofcAMP-alkyneCC(lanes 3 and 4) or 5 µMof pre-clicked cAMP-alkyne-azide-biotin(lanes 5 and 6). Bar plot displays intensity of biotin (panel A),PKARI (panel B) and normalized biotin intensity to PKARI incorrespondingsample(panelC).


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Introducing a non-natural stable molecule with fluorescence properties such as TAMRA forlabeling/capturingincomplexmediareducesthebackgroundoriginatedbybiotin.

Recombinant PKARI was labeled as previously established using the optimal determinedconditions: 5µMcAMP-alkyne capture compound (CC2), 100µMCuSO4, 200µMBTTE (CC18), 5mM sodium ascorbate and 10 µM TAMRA-azide (CC 5). Aliquots of samples were taken out atdifferenttimepointsduringthecourseoftheCuAACreactionandanalyzedbySDS-PAGE/W.Blot.

Figure2-10,showsthatusingthenewTAMRA-azide iscompatibletotheCuAACreactionfor

PKARI labeling. The TAMRA label is observable only after 15 min of reaction and the intensitycontinuesto increaseuntil60minofCuAACreactionalwayswiththeexpectedcompetitioneffect,wheretheintensityislowerinthesamplespre-incubatedwithexcesscAMP.Infact,theresultsalsoshowthattheproteinintegrityisnotaffectedduringtheCuAACreactionusingtheTAMRA-azideandthe described conditions, as it was previously observedwhen running the equivalent time courseexperimentwiththebiotin-azidecounterpart(seeFigure2-6,onpage35).

ThisapproachusingtheTAMRAlabelshouldbeusedforfutureexperimentsincomplexmediasuchascellularlysateswheretheclickreactionefficiencycanbemoreeasilydeterminedwithouttheconfoundingbackgroundofbiotinylatedproteinswhenusing thebiotin-azide for theCuAAC.Aftertheparametersforthereactionaredeterminedforthespecifictargetbeingevaluatedinaparticularlysate,thebiotinclickwillhavetobeusedforthecaptureandenrichmentofthetargetsbecauseoftheavailablestreptavidin-magneticbeadssystem.

Figure 2-10: Effect of timeof CuAAC reaction on the labeling ofPKARI with a cAMP-alkyne capture compound and a TAMRA-azide.PKARI was pre-incubated with (C) or without (A) excess freecAMP. The samples were then incubated with 5 µM of cAMP-alkyneCC,10µMTAMRA-azide,200µMCuSO4,400µMBTTEand5mM sodium ascorbate. Aliquotswere taken at 15, 30 and 60min of CuAAC reaction. Bar plot displays intensity of TAMRA(panel A), PKARI (panel B) and normalized TAMRA intensity toPKARIincorrespondingsample(panelC).


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2.2.2. Mass Spectrometry analysis of captured proteins fromHEK293 andHepG2lysatesusingaclassicalandclickablecAMPcapturecompound

Withalloptimization stepsput inplace,wesetout to findwhetherPKARwasenrichedandcapturedfromacellularlysateusingCuAACreaction.

Inan initialexperimentdifferent lysisbufferswereusedtosolubilizecellularpellets.HEK293cellswere lysed inabuffercontainingornotn-Dodecylβ-D-maltoside (DDM).Alternatively, ready-madeHepG2lysateswereobtainedfromInVivo.ThedifferentlysateswereincubatedwiththecAMPcapture compounds in optimized conditions as previously described in the labeling experiments.After the CuAAC reaction proteins were either precipitated or not to remove excess non-reactedbiotin-azide and re-solubilized before incubation with the streptavidin-coated magnetic beads. Adetailed description of themethod can be found in 2.3.4 Click capture of cAMP-binding proteinsfromcelllysates,onpage53.

Table2–1gathersthedataobtainedfromtheproteomicsanalysisoftheexperiment.

ClassicalcAMP-CC

ClickablecAMP-CC(noprecipitation)

ClickablecAMP-CC(withprecipitation)

Assay Competition Assay Competition Assay Competition

Totalproteinsidentified(LFQ>0)

• HEK293(+DDM) 901 1354 640 1448 1435 1467

• HEK293(-DDM) 1004 1045 991 1333 1394 1389

• HepG2 892 1256 1417 345 1459 1545

Assay/Competition fold-change > 2(%oftotalproteinsinAssay) 24 15 2

• HEK293(+DDM)170

(18.9%)98

(15.3%)124

(8.6%)

• HEK293(-DDM) 209(20.8%)

189(19.1%)

163(11.7%)

• HepG2 118(13.2%)

1226(86.5%)

178(12.2%)

Assay/Competition fold-change > 2acrossthedifferentworkflows 0

• HEK293(+DDM) 5

• HEK293(-DDM) 1

• HepG2 3Table2–1:SummaryofidentificationsfromcAMPcaptureexperimentsinHEK293andHepG2lysates.ListofproteinswasobtainedusingMaxQuantv1.2.2.4.Numbersforpositiveproteinidentificationsineachsamplearegivenandintensity-basedcalculated fold-changesbetweenassaysandcorrespondingcompetitionsaswellasacrossall sampleswithineach lysatearealsopresented.


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Table2–1,onpage42, shows thatusingdifferent lysisbuffers to solubilize theproteomeofthe samecell linewillproducedifferent resultsnotonly in specifically competedproteins, rangingfrom 98 up to 209 depending on the capturemethod, but also in the total of identified proteinswhich was of only 640 in the click-capture without precipitation method and as high as 1467differentproteins inthecompetitionsampleoftheclick-capturefollowedbyprecipitationmethod.In the commercial HepG2 lysate there was a low value of 345 identified proteins in one of thesampleswhichbydefaultgreatlyincreasesthecalculatednumberofspecificallycompetedproteins,but that could have been an experimental error.When the calculated competed proteins in eachlysate subset are compared between them according to the capture method, 24 proteins areidentifiedascommonlycompetedfortheclassicalcapturecompoundacrossthedifferentlysates,15for the clickable cAMP capture compound and only 2 when an additional step of proteinprecipitationisaddedtotheclickablecapturecompound.Theseproteinsarenotcommonacrossallsamplesandmethodsbuttherearetwoproteinsthatarecompetedinalllysatesidentifiedbothwiththe classical capture compound and the clickable cAMP capture compound without proteinprecipitation–RPS8,aribosomalproteinandSRRM2,aproteininvolvedinpre-mRNAsplicing.

The different methods utilized in the experiment did not successfully produce a commonspecific binder across all proteomes and capture methods although some commonly competedproteinswere identified across the differentmethodswithin one type of lysate. These commonlycompeted proteins were mostly ribosome-associated proteins, which are usually consideredunspecificbackground(Table2–2).

Table 2–2: Proteins identified asspecifically competed with cAMPacross the different capturemethods in each type of cellularlysate.

ProteinGeneName HEK293(+DDM) HEK293(-DDM) HepG2

H1F3 ✓

RPS19 ✓

RPL27A ✓ ✓

RPS18 ✓

HABP1 ✓

SERPINB6 ✓

HRNPG ✓

RPL21 ✓


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Comparing the competed proteins between each lysate within the same capture methodranges between 37 and 54 for the classical cAMP capture compound, 20 and 82 for the clickablecAMPcapturecompoundwithouttheproteinprecipitationstepandwhenthisproteinprecipitationstep is introduced it ranges between 18 and 25. These values are represented in Table 2–3highlightedinbold. It is interestingtonoticethatthenumberofcommonly identifiedascompetedproteinsusingtheclickablecAMPcapturecompoundwithaprecipitationstepisgreatlyreducedincomparisontotheothertwomethodsbuttheoverallnumberof identifiedproteins issimilar.Alsohighlighted in Table 2–3 in italic are thenumberof commonly identifiedproteins as competedbycAMPfromthesamelysatebutusingdifferentcapturemethods.

ClassicalcAMP-CC



HEK293(+DDM)

HEK293(-DDM) HepG2 HEK293

(+DDM)HEK293(-DDM) HepG2 HEK293

(+DDM)HEK293(-DDM) HepG2

ClassicalcAMP-CC

HEK293(+DDM)

HEK293(-DDM) 54

HepG2 49 37


HEK293(+DDM) 42 25 31

HEK293(-DDM) 37 20 19 20

HepG2 118 140 68 55 82

ClickablecAMP-CC(precipitation)

HEK293(+DDM) 18 18 7 8 14 70

HEK293(-DDM) 14 15 7 6 10 103 25

HepG2 25 22 5 8 15 102 18 24

Table2–3:NumberofproteinsidentifiedascompetedbycAMPincommonbetweenthedifferentsamples.Highlightedvaluesinboldrepresentthecompetedproteinsbetweendifferentlysatesusingthesamecapturemethod;highlightedvaluesinitalicrepresentthenumberofproteinsfromoneparticularlysatecompetedacrossthedifferentcapturemethods.


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Focusing on the results obtained for PKA, Table 2–4 compiles the calculated fold-change ofassayandcompetitionintensitiesandthenumberofMSspectraobtainedforeachidentifiedsubunitofPKA(threeregulatorysubunits–PRKAR1A,PRKAR2AandPRKAR2B–andonecatalyticsubunit–PKACA).

Protein

ClassicalfullcAMP-CC ClickablecAMP-CC(noprecipitation)


Genename Intensityfold-

change(log2)

MSCounts Intensityfold-

change(log2)

MSCounts Intensityfold-

change(log2)

MSCounts

(Assay) (Comp.) (Assay) (Comp.) (Assay) (Comp.)

PRKAR1A

• HEK293(+DDM) 14.4 10 0 0.0 0 0 0.0 1 0

• HEK293(-DDM) 7.8 9 0 0.1 0 0 -0.1 0 1

• HepG2 13.9 10 0 -8.9 0 0 2.8 2 1

PRKAR2A

• HEK293(+DDM) 6.9 12 0 4.1 0 0 0.9 2 1

• HEK293(-DDM) 6.7 10 0 -0.9 0 0 0.9 2 0

• HepG2 8.7 16 0 7.7 0 0 -0.3 4 2

PRKAR2B

• HEK293(+DDM) 8.2 1 0 0.0 0 0 0.0 0 0

• HEK293(-DDM) 8.4 2 0 0.0 0 0 0.0 0 0

• HepG2 8.2 3 0 0.0 0 0 0.0 0 0

PKACA

• HEK293(+DDM) 0.0 0 0 0.0 0 0 7.8 0 0

• HEK293(-DDM) 0.0 0 0 -7.9 0 0 0.0 0 0

• HepG2 0.0 0 0 9.1 2 0 0.0 0 0

Table2–4:Calculatedintensityfold-changes(assayvs.competition)andMScountsforidentifiedPKAsubunitsineachsample.

As seen on Table 2–4, the classical full cAMP capture compound specifically captures the

regulatory subunitsofPKAacrossall typesof cell lysate, ranging from6.7 to14.4 log2 fold-changebetweentheassayandthecorrespondingcompetition.Additionally,noMSspectrawereobtainedinthe competition samples using the classical full capture compound, confirming the successfulblockageofthecapturecompoundbyfreecAMPcompetitor.


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Resultsontheclickapproacharemorevariable.Table2–4,onpage45,showsthatthereisabigreductiononthenumberofidentifiedMSspectrainassaysusingtheclickablecapturecompoundwhen compared to the classical full capture compound,meaning that there is little enrichmentofthese subunits of PKA. Althoughwith only fewMS spectra, and sometimes none, PKA regulatorysubunit 2A (PRKAR2A) is successfully identified as competed using the new developed CuAACapproach in both HEK293 and HepG2 cells with or without protein precipitation. Additionally,PRKAR1AisalsospecificallyidentifiedwithpositiveMSspectrainHepG2lysatewhenusingaproteinprecipitationstepaftertheCuAACreactionwiththeclickablecAMPcapturecompound.

Fromanoverallobservationof the resultspresented inTable2–4,eitherHEK293cells lysedwith a DDM-containing buffer or HepG2 lysate obtained externally can be used for furtherexperimentstoimprovethemethodofcapturingcAMP-bindingproteinsincellularlysatesusingtheCuAACreaction.Noexactdataonefficiencyorreproducibilityofthisnewmethodcouldbeextractedfrom this experiment due to its design lacking technical replicates; therefore, a new captureexperimentofcAMP-bindingproteinsusingCuAACreactionwasperformed.

HEK293cells lysedinabuffercontainingDDMwereincubatedwitheitherclassicalfullcAMPcapturecompoundorwithclickablecAMP-alkynecapturecompound.Tocorrespondingcompetitionsamples free cAMP was added prior to the capture compounds. Additionally, control samplescontaining no capture compound were processed in parallel. After photo-crosslinking and CuAACreactionallsamplesweresubmittedtomethanolprecipitationandreconstitutedbeforeincubationwith streptavidin-coated magnetic beads. All samples were processed in technical triplicates toestimatereproducibilityandcalculatesignificanceofidentifications.

Table2–5belowpresentsthesummaryofidentificationsandcalculatedspecificandsignificantproteinsinacaptureexperimentusingcAMPcapturecompoundsinHEK293lysate.

ClassicalcAMP-CC ClickablecAMP-CC Assay Competition Assay Competition

Totalproteinsidentified 1462 1429 1591 1637

Student’st-test<0.05(%oftotalproteinsinAssay)

27(1.8%)

50(3.1%)

Assay/Competitionfold-change>2(%oftotalproteinsinAssay)

382(26.1%)

177(11.1%)

Competedandsignificantproteins(%oftotal)

21(1.4%)

17(1.1%)

Table2–5:SummaryofidentificationsfromcAMPcaptureexperimentsinHEK293lysates.ListofproteinswasobtainedusingMaxQuantv1.2.2.4.Numbersforpositiveproteinidentificationsineachsamplearegivenandintensity-basedcalculatedfold-changesbetweenassaysandcorrespondingcompetitions.

Having replicate samples allows for the calculation of the significance of the calculated

intensity fold-change between assays and competitions. This statistical analysis method narrowsdown382potentialcompetedhitstoonly21with95%confidenceinthecaptureexperimentsusingthe classical full cAMP capture compound. In the CuAAC reactionmethod using the cAMP-alkynecompoundthepotentialhitswithsignificantvaluesare17intotal(Table2–5).


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Inanon-biasedapproach the listof significantly competedproteins forbothsetsof capturecompoundswouldbepresented,butthepurposeofthisexperimentdesignistofocusonthecAMP-bindingproteinPKA.TheresultsfocusedonPKAsubunitsarepresentedinTable2–6.

Protein

Genename Intensityfold-change(log2)

Student’st-test(pvalue)

SumofSpectralCounts

(Assay) (Comp.)

PRKAR1A

• fullcAMP-CC 13.1 0.048 5 0

• clickablecAMP-CC -0.1 0.932 2 1

PRKAR2A

• fullcAMP-CC 2.4 0.027 5 0

• clickablecAMP-CC -0.1 0.927 2 4

PRKAR2B

• fullcAMP-CC - - 2 0

• clickablecAMP-CC - - 0 0

PRKACA

• fullcAMP-CC - - 0 0

• clickablecAMP-CC -11.1 0.192 2 4Table2–6:Calculatedintensityfold-changes(assayvs.competition)andcorrespondingsignificancevalueforidentifiedPKAsubunitsfromcaptureexperimentsusingfullandclickablecAMPcapturecompounds.Sumofspectralcountsoftriplicateassaysandcompetitionsisalsoshown.

The classical full cAMP capture compound was able to specifically capture two different

regulatory subunits of PKA – PRKAR1A and PRKAR2A – from the HEK293 lysate. Intensity fold-changesof13.1and2.4with5identifiedMSspectraforbothregulatorysubunitswere,respectively,obtained(Table2–6).

ThemethodusedwiththeclickablecAMPcapturecompoundwasabletoproducemoreMSspectraforPRKAR1Aintheassayscomparedtothecontrols,butthecorrespondingintensitieswhenfold-changeswerecalculatedgiveresulttoanegativevalue(-0.1)althoughnon-significant(pvalue=0.932).ForPRKAR2A,moreMSspectrawere identified in thecompetitionsamplesof theclickablecapturecompoundandintensityfold-changewasalsonegative(Table2–6).Thelackofsignificanceof these results does not allow any conclusive analysis on the performance of themethod to bemade.

Previousexperimentspresentedinthischapterhaveshownalackofreproducibilityregardingthe competition effect expected when excess free cAMP competitor is added to the samples (asexample, see Figure 2-7 and Figure 2-9, on pages 37 and 40, respectively). Even if this lack ofcompetitionwouldbefurtherinvestigatedandresolved,thenaturalhighconcentrationofcAMPinacellularenvironmentisnottheoptimalsystemtofurtherproceedwithcapturingtargetsinanintactcellenvironment.

A non-natural selectivity function such as the drug dasatinib was chosen for subsequentexperiments.

Chapter2:Labelingaproteinusingclickchemistry|SpecificMethods

48

2.3. SPECIFICMETHODSFORTHECAMP/PKARISYSTEM2.3.1. StructuresofusedcompoundsCC1:ClassicalfullcAMPcapturecompound

CC2:ClickablecAMPalkynecapturecompound(containsselectivityandphotoreactivefunctions)

CC3:Clickablebiotin-PEG-azide(containslabelingfunction)

CC4:ClickedcAMP-biotincapturecompound.ReactionproductofCC2andCC3


49

CC5:ClickableTAMRA[tetramethylrhodamine]azide(containslabelingfunction)

CC13:cAMPcompetitor

CC16:Copper-chelatingligandTBTATris(benzyltriazolylmethyl)amine

CC17:Copper-chelatingligandTHPTATris(3-hydroxypropyltriazolylmethyl)amine

CC18:Copper-chelatingligandBTTEbis(tert-butyltriazoly)ethanol


50

2.3.2. Establishingandoptimizingclick-captureofPKARIusingacAMPCC16pmolof recombinanthumanPKARIwaspipetted into200µLPCR-tubesanddilutedwith

CaptureBufferandwatertoafinalconcentrationof0.1µM.Tothecompetitionsamples,1,2or4mMofcAMP(CC13)wasaddedand incubatedat least10minon ice.Next, cAMP-alkynecapturecompound(CC2)wasaddedtothesamplesatdifferentconcentrationsrangingfrom0.31µMto10µM.Thesampleswereincubatedatleast30minrotatingat4°CandthenUV-irradiatedfor10minat310 nm in a CaproBox™ to promote photo crosslinking. To the samples containing cAMP-alkyne,reagentsfortheCuAACreactionwereaddedandtestedatdifferentconcentrations:biotin-azide(CC3)waspresentbetween5µMand30µM;CuSO4between50µMand750µM;andthreedifferentcopperligandsusedindividually–TBTA(CC16)at250µM,THPTA(CC17)at150µMor250µM,orBTTE(CC18)usedat150µMor200µM.Toinitiatetheclickreaction,sodiumascorbatewasalwaysadded lastly at a final concentration of 2.5 mM. The CuAAC time of reaction was performed atdifferent times. A new TAMRA-labeled azide (CC 5) was also clicked to the cAMP-alkyne capturecompound in one set of samples at a final concentration of 5 µM.All parameters tested for eachexperimentandcorrespondingobtainedresultsarespecifiedinTable2–7onthefollowing2pages.

Insomeexperiments,paralleltotheclicksamples,thefullclassicalcAMPcapturecompound(CC 1) was added at a final concentration of 5 µM to be used as a reference control. To thesecontrols,allclickreagentsexceptfortheazidewereadded.

TheresultswerevisualizedbySDS-PAGEfollowedbyBlottingwithchemiluminescencereadoutonx-rayfilmusingeitherastreptavidin-HRPsolution(forbiotin-labeledsamples)orIgGanti-TAMRAfollowedbyanti-IgG-HRPsecondaryantibody(forTAMRA-labeledsamples).

BandintensitywasmeasuredusingImageJsoftwareforfold-changecalculations.

2.3.3. PreparationofHEK293lysatesHEK293 cell pellets were lysed in 2X their volume with a hypotonic buffer (10 mM HEPES

pH7.9, 1.5 mM MgCl2, 10 mM KCl) containing (+) or not (-) 0.5%(w/v) DDM. Both buffers weresupplementedwithproteinaseinhibitorsandbenzonaseatthetimeoflysis.After30minincubationon ice, the lysatewasdounced20Xusinga tightpestleandthenrunthrougha21Gneedlewithasyringe.Remainingdebriswaspelletedbyrefrigeratedcentrifugationat15.000rpmfor30min.Thesupernatantwas recovered and the leftover pelletwas discarded. The lysateswere aliquoted andsnap-frozeninliquidnitrogen.Sampleswerestoredat-80°Cuntiluse.

ProteinconcentrationoflysateswasdeterminedbytheBCAassayasdescribedintheGeneralMethodssection(6.2.1Proteinconcentrationdetermination,onpage118).


51

PARAMETEROPTIMIZATIONFORLABELINGANDCAPTURINGOFPKARIUSINGCLICKCHEMISTRY cAMP-alkyne

CCcAMP

competitorCuAACreaction

biotin-azide TAMRA-azide CuSO4Copperligand sodium

ascorbate TBTA THPTA BTTE

Experiment1

Results2.2.1.1

(Figure2-2)

5µM -

30min 10µM - 50µM 250µM - - 2.5mM2.5µM -1µM -0.5µM -0µM -

Experiment2

Results2.2.1.1

(Figure2-3)

5µM

1mM 30min 10µM - 250µM 250µM - - 2.5mM2.5µM1.25µM0.63µM0.31µM

Experiment3

Results2.2.1.2

(Figure2-4)

5µM - 30min 10µM -

0µM

250µM - - 2.5mM50µM250µM500µM750µM

Experiment4

Results2.2.1.2

(Figure2-5)

5µM 2mM 30min 10µM -250µM

0µM250µM - - 2.5mM

50µM 250µM

Experiment5

Results2.2.1.3

(Figure2-6)

5µM -

2min

10µM - 250µM - 250µM - 2.5mM

10min20min30min60min90min120min180min


52

PARAMETEROPTIMIZATIONFORLABELINGANDCAPTURINGOFPKARIUSINGCLICKCHEMISTRY cAMP-alkyne

CCcAMP

competitorCuAACreaction

biotin-azide TAMRA-azide CuSO4Copperligand sodium

ascorbate TBTA THPTA BTTE

Experiment6

Results2.2.1.4

(Figure2-7)

5µM 2mM 30min

0µM

- 250µM - 250µM - 2.5mM5µM10µM20µM30µM

Experiment7

Results2.2.1.5

(Figure2-8)

5µM 2mM 20min 10µM - 50µM - 150µM - 2.5mM

Experiment8

Results2.2.1.6

(Figure2-9)

5µM 2mM 10min 10µM - 50µM - - 150µM 2.5mM

Experiment9

Results2.2.1.7

(Figure2-10)

5µM 2mM

15min

- 5µM 100µM - - 200µM 2.5mM30min

60min

Experiment10

Results2.2.2

10µM 4mM 30min 20µM - 100µM - - 200µM 2.5mM

Table2–7:SummaryofparametersutilizedinPKARIlabelingandcapturingwithacAMPcapturecompoundviaaCuAACreaction


53

2.3.4. ClickcaptureofcAMP-bindingproteinsfromcelllysatesHEK293andHepG2cellularlysateswerepre-clearedofendogenouslybiotinylatedproteinsby

incubating with streptavidin-coated magnetic beads prior to the capture experiment. The lysates

wereincubatedwiththebeadsfor45minrotatingat4°Candthenthesewereremovedusingthe

caproMag™.

The cleared lysatewas distributed in 200µL PCR-tube strips and dilutedwithwater and 5X

Capture Buffer. To the competition samples, 4 mM of cAMP (CC 13) was added and the

correspondingvolumeofwaterwasaddedtotheassaysamples.Thevialswereincubatedfor10min

oniceandthen10µMofeithercAMP-alkyne(CC2)orclassicalfullcAMP(CC1)capturecompounds

wereaddedtothedesignatedtubes.Thesampleswere incubatedfor1hrotatingat4°Candthen

irradiatedfor10minat310nminacaproBox™topromotephoto-crosslinkingofthecompoundsto

thebindingtargets.TothevialscontainingthecAMP-alkynecapturecompoundtheCuAACreaction

wasperformedbyadding20µMbiotin-azide (CC3),100µMCuSO4,200µMBTTE (CC18)and2.5

mMsodiumascorbate.TothevialscontainingtheclassicalfullcAMPcapturecompound,thesame

volumeofwaterwasadded.Theclickreactionranfor30minshakingatroomtemperature.

Theclickedsamplesweredividedintwoandonesetwasmixedwithmethanolpre-cooledat-

20 °C to precipitate proteins and leave non-clicked biotin-azide in solution. These samples were

storedover-nightat-80°Ctoallowforprecipitation.Theotherfractionofclickedsamplesandthe

samplescontainingtheclassicalfullcapturecompoundweresnap-frozeninliquidnitrogenandkept

at-80°Cforthedurationoftheprecipitationprocedure.Theprecipitatedsampleswerepelletedfor

20min spinning at 15.000 rpm. The supernatantwas discarded and the tubes let to dry at room

temperature.Theprecipitatewaswashedthreetimesbyressuspendingitinfreshcoldmethanoland

vigorouslyvortexingtohomogenizethepellet.Afterthelastwash,thepelletswereresuspendedin

4%SDSandsonicateduntilaclearsolutionwasobtained.ThesamplesweredilutedwithPBS toa

finalconcentrationof0.4%SDSandincubatedwithstreptavidin-coatedmagneticbeadsforcapturing

of biotinylated proteins. The non-precipitated samples were thawed and also incubated with the

beadsforcapturing.

Themagneticbeadswereincubatedwiththelysatesforatleast1hrotatingat4°Candthen

recoveredusingacaproMag™.Thebeadswerethenwashedsixtimeswithwashbufferfollowedby

threetimeswashwith80%acetonitrileandafinalwashwithMS-gradewater.

0.5µgoftrypsininammoniumbicarbonatewasaddedtothebeadsandincubatedover-night

shakingat37°C.Thesupernatantcontainingthetrypticdigestwasrecoveredtocleantubes;next,

the beads were washed with MS-grade water and the wash solution pooled with the original

supernatant. All samples were lyophilized and analyzed by LC-MS/MS following the protocol

describedintheGeneralMethodssection(6.3MassSpectrometry,onpage121).

Chapter3:IdentifyingDasatinibtargetsincells|Introduction

54

3. IdentifyingDasatinibtargetsincells3.1. INTRODUCTION

Having implementedthebasicstepsforanovelclick-capturesysteminacell lysatewiththe

cAMPcapturecompound,thenextstepwouldbetoproceedtoalivingcellcapture.cAMPisanon-

permeablehighlyabundantmoleculeincells, inthemicromolarrange,whichmakesitnon-idealto

establishanewworkflowforalivingcellcapturingsystem.Ideally,thecapturecompoundtobeused

for target identification in cell culture should be a non-naturally occurringmolecule.With this in

mind,acollaborationwasestablishedwithProf.YaofromtheUniversityofSingaporewhopublished

apaperonthecellulartargetsofdasatinibusingaclickablecapturecompound92.

Dasatinib (CC 14) is a protein tyrosine kinase inhibitor initially developed by Bristol-Meyers

Squibb(commercializedasSprycel)totreatimatinib-resistantchronicmyeloidleukemia93.

Chronicmyeloidleukemia(CML)accountsfor15%ofallnewcasesofleukemiaintheWestern

hemispherewithaprevalenceof1/100.000peryear,whichuntreatedhasamediansurvivalof5to7

years94. InCML thehematopoietic stemcells possess an abnormal chromosome, thePhiladelphia

chromosome,whichoriginates fromthereciprocal translocationofaregionofchromosome9that

containstheAblesononcogene(ABL)andthebreakclusterregion(BCR)genefromchromosome22.

This translocation produces a new fusion gene that ends up being translated into the BCR-ABL

protein.TheABLfamilyofproteintyrosinekinases,whichiscomprisedofABL1andABL2(alsoknown

as ARG), link diverse extracellular stimuli to signaling pathways that control cell growth, survival,

invasion,adhesionandmigration.Thereisastrongrelationshipbetweenderegulationofthisfamily

of protein kinases and cancer, from leukemia to solid tumors95. Whilst in most leukemia the

deregulation originates from chromosome translocations that produce fusion proteins, in solid

tumors the activation of the ABL kinases is driven by their over-expression, chemokine receptors,

oxidativestressand/orinactivationofnegativeregulatoryproteins95.

ABL1 and ABL2 function redundantly in some cellular contexts, but also have unique roles,

owing to distinct and conserved sequences and structural domains (Figure 3-1, onpage 55).ABL1encodesanon-receptortyrosinekinasethatphosphorylatessubstrateproteinswithcrucialcellular

activities such as increased proliferation, loss of stromal adhesion, and resistance to apoptosis94.

Abl1-knockout mice exhibit diverse abnormalities leading to premature senescence and neonatal

morbidity.Bycontrast,Abl2-knockoutmiceareviablethoughtheyexhibitneuronaldefects96.

UnlikePKAwithitssingleactivatingmoleculecAMP(asdiscussedinthepreviouschapter),the

ABLkinaseisactivatedbymanydifferentextrinsicligands,suchasepidermalgrowthfactorreceptor

(EGFR),platelet-derivedgrowthfactorreceptor(PDGFR),vascularendothelialgrowthfactorreceptor

(VEGFR),andothers95.TheABLkinaseisalsoactivatedbyintrinsicsignalssuchasDNAdamageand

oxidativestress97.


55

Figure3-1:SchematicrepresentationofthemodulardomainsoftheABLkinases.Alternative splicing of ABL1 and ABL2 produces several isoforms, including the 1a isoforms (as shown by the straight line) and the 1bisoforms (jagged line); the 1b isoforms are targeted for amino-terminal myristoylation. The ABL N termini are comprised of the SRChomology 3 (SH3), SH2 and SH1 (tyrosine kinase) domains. The ABL carboxyl termini contain a conserved filamentous (F)-actin-bindingdomain(BD).ABL1hasaglobular(G)-actin-bindingdomainandaDNA-bindingdomain,whereasABL2hasasecondinternalF-actin-bindingdomainandamicrotubule(MT)-bindingdomain.ABL1hasthreenuclearlocalizationsignal(NLS)motifsandonenuclearexportsignal(NES)initsCterminus.BothABL1andABL2haveconservedPXXPmotifstomediateprotein–proteininteractions.Phosphorylation(P)ofABL1atY412withintheactivationloop(Y439inABL2)andY245intheSH2-kinasedomainlinker(Y272inABL2)stabilizestheactiveconformation.ReproducedfromGreuberetal.95

The catalytic activities of the ABL kinases are tightly regulated by inter and intramolecular

interactions, and post-translational modifications (Figure 3-2). The intramolecular inhibition is

obtained by the formation of a clamp structure involving a SRC homology (SH) 3 domain*, a SH2

domainandaSH1domain.ABLactivitycanbenegativelyorpositivelyregulatedby intermolecular

interactionswith distinct binding partners. In general, the intermolecular interactions that disrupt

the autoinhibitory interactions and stabilize the active (open) conformation of the ABL kinases

promoteincreasedenzymaticactivity.Forexample,thecatalyticefficiencyofABLcanbeenhanced

byinteractionswithadaptorproteinssuchasRasandRabinteractor1(RIN1),whichinteractsboth

withtheSH3andSH2domains95.

Figure3-2:Representationofauto-inhibited(closed)andactive(open)ABLkinases.IntramolecularinteractionsplayaroleintheregulationofABLkinaseactivity.TheSH3domainbindstothelinkersequenceconnectingtheSH2andthekinasedomains,andtheSH2domain interactswithC-terminal lobeof thekinase(SH1)domainforminganSH3–SH2clampstructure.ThemyristoylatedresidueintheNterminusofthe1bABLisoformsbindstoahydrophobicpocketwithintheC-lobeofthekinasedomain,stabilizingtheauto-inhibitedconformation.TheconfigurationandinteractionsoftheABLC-terminalsequencesarenotincludedinthemodel.DashedlinerepresentsABLN-terminalsequencesupstreamoftheSH3domainthatfoldovertopromotebindingofthemyristoylgrouptoapocketintheC-lobeofthekinasedomain.AdaptedfromGreuberetal.95

*SRChomology(SH)domainsaredividedinthreecategories:SH1domainreferstothetyrosinekinasedomainwhichwas

firstidentifiedintheSRCkinase;SH2domainisaproteinmodulethatbindstotyrosinephosphorylatedsitesinasequence-

specificcontext;SH3domainisaproteinmodulethatbindstoproline-richsequences.


56

Among the proteins reported to negatively regulate ABL kinase activity are peroxiredoxin 1

(PRDX1;alsoknownasPAG)98,thetumorsuppressorproteinFUS1(alsoknownasTUSC2)

99andthe

phosphatase-interacting adaptor protein PSTPIP1100. Finally, the enzymatic activity of the ABL

kinasesisalsoregulatedbytyrosinephosphorylation,whichstabilizestheactiveconformation,and

canoccurbytrans-phosphorylationbetweenABLkinasesormaybemediatedbySRCfamilykinases95.

Inhibitors of the ABL kinases are classified into three main classes on the basis of their

mechanism of action: type 1 inhibitors target the active conformation of the kinase domain

(dasatinibandbosutinib)101,102

,whereastype2 inhibitorsstabilizetheinactiveconformationofthe

kinasedomain,preventingitsactivation(imatinib,nilotinibandponatinib)103–105

.Thethirdcategory

of inhibitors includesallosteric inhibitors,whichdonot compete forATPbindingbutwhich rather

bindtoregulatorydomainstoinhibitkinaseactivity(GNF-2andGNF-5)106,107

.IncontrasttotheATP-

competitiveinhibitorsthattargetmultiplekinases,theallostericinhibitorsarehighlyselectiveforthe

ABLkinases95.

In2011,Davisetal.108publishedanextensiveanalysisontheselectivityof72kinaseinhibitorsagainst442kinases,bymeansofacompetitionbindingassay.Fromthose72kinaseinhibitors,only7

presentedacalculatedmedianaffinityoflessthan30nMforthetestedvariationsofABL1andABL2:

imatinib103,dasatinib

109,bosutinib

102,nilotinib

104,VX-680/MK-0457

110,PD-173955

111andAST-487

112.Allthesekinaseinhibitorsinteractedwithotherproteinsatthesame30nMcutoff,rangingfrom

only2morewithnilotinibto43additionaltargetswithAST-487.Basedonthesupplementalresults

fromDavisetal. 108, Figure3-3wasproduced to illustrate theproteins that are targetedby theseselectedkinaseinhibitorsandhowtheyoverlapbetweenthem.

Figure3-3:Proteinsinhibitedbykinaseinhibitorswithanaffinityoflessthan30nMforABL1andABL2.Sevenkinaseinhibitors(imatinib,dasatinib,VX-680/MK-0457,bosutinib,nilotinib,PD-173955andAST-487)fromatotalof72presentedanaffinityagainstABL1andABL2oflessthan30nM.Thisthresholdwasappliedtotheothertargetswithpositiveresultsandlisted.Amapoftheoverlappingtargetsbyeachofthekinaseinhibitorswasdrawn.ThisfigureisbasedresultsfromDavisetal.108


57

Dasatinib is an ATP-competitive protein tyrosine kinase inhibitor, which was originally

identifiedasapotentinhibitorofSRCfamilykinases(includingSRC,LCK,HCK,YES,FGR,LYNandFYN)

andwas subsequently found to have activity against ABL, KIT, themacrophage colony stimulating

factorreceptor(FMS),theplatelet-derivedgrowthfactorreceptor(PDGFR)andtheephrinreceptor

family members EPHB1, EPHB2 and EPHB4113. The receptor tyrosine kinase DDR1 and the

oxireductaseNQO2havealsobeenrecently identifiedasspecificbindersofdasatinib19. Theexact

mechanismofdasatinibactioninCMLisstillunknownandtherearemanytheories114.Somestudies

have shown that dasatinib induces defects in spindle formation, cell cycle arrest, and centrosome

alterations in leukemic cells, tumor cell lines, as well as in normal cells115. The mechanisms of

dasatinib and its role in CML as well as how to decrease the toxicity of dasatinib require further

investigation114,soanunbiasedfullproteomeanalysisofdasatinibbindersmightelucidatesomeof

thosemechanisms.

In2012,Shietal.92characterizedtheproteomeofdasatinibtargetsbyuseofclickablecapture

compounds. The uniqueness of this approachwas to address those targets directly in living cells,

where theprotein-ligand, aswell asnativeprotein-protein interactionswouldbemorenatural, as

opposed to potential artifacts and alternative protein conformations created during cell lysate

preparations that are commonly used in binding/inhibition assays. Although they identified some

protein andnon-protein kinases in livingK562 cells*, noneof themwere theABLor SRC familyof

proteins;thesewereonlyidentifiedwhenthecapturewasperformedinlysate(supplementarydata

tables from Shi et al. 92). Also lacking in this study is a parallel competition assay to correctly

determinespecificbindersofdasatinib.

BasedontheworkfromShietal.92andhavingadirectcollaborationwiththatgroup,wesetout to capture dasatinib targets in living cells using our established and optimized click capture

workflowdescribedinthepreviouschapter.

*K562cells:celllinecontainingthePhiladelphiachromosomeandthereforecarryingtheBCR-ABLfusionprotein.Usedas

cellmodelofchronicmyeloidleukemia(CML)

Chapter3:IdentifyingDasatinibtargetsincells|Results

58

3.2. RESULTS3.2.1. Introducinganewcopper-chelatingazideintheCuAACreaction

The experiments done so far to optimize the CuAAC reaction were all performed using a

commerciallyavailablebiotin-azide (CC3,onpage122)asdescribed in thepreviouschapter (2.2.1

Establishingandoptimizingclick-captureofPKARIusingacAMPCC,onpage31).

Valentina Bevilacqua, a fellow member of the BioChemLig consortium, synthesized new

copper-chelating azides to be used in bioconjugation experiments116. The most efficient azide

regardingreactionkineticsatthetimewaschosenandabiotin(CC7)orTAMRA(CC8)anchorswere

attached to it to perform labeling and capture experiments. The structures of the compounds are

illustratedontheSpecificMethodssection(3.3.1Structuresofusedcompounds),onpage77.

An initialexperimentwasdesigned to test theefficiencyof thisnewazide in the labelingof

recombinant SRC protein solution or in K562 cells lysate using a clickable dasatinib capture

compound (CC 6). The previously optimized click reaction parameters with the cAMP capture

compound were used as reference and the conditions used to measure the reaction kinetics in

Bevilacqua’s system were also used. The parameters used in the reaction kinetics experiments

seemedfromthestarttobetooharshforabiologicalsystem;hence,anewsetofparameterswas

designedandincludedinthelabelingexperiment.

Figure 3-4: Labeling of recombinant SRC and K562 cell lysatewith a clickable dasatinib-alkyne capture compound using a new copper-chelatingazidewithaTAMRAlabel.Recombinant SRC (lanes 1 – 6) or K562 cell lysate (lanes 7 – 12)were pre-incubatedwith (C) orwithout (A) excess free dasatinib. Thesampleswerethenincubatedwithdasatinib-alkyneCCandclickedwithclassicalbiotin-azideandBTTEH(lanes1,2,7and8),Bevilaqua’sinitialconditionswiththecopper-chelatingazide(lanes3,4,9and10)andanewoptimizedCuAACreactionwiththenewcopper-chelatingazide(lanes5,6,11and12).BarplotdisplaysintensityofTAMRAfromthebandswithinthedrawnbox.


59

AsseenonFigure3-4,onpage58,thepreviouslyestablishedCuAACreactionconditionsshow

the faintestsignalwhencomparedtotheothersamples (Figure3-4, in-houseCuAAC lanes inboth

recombinantSrc-GSTandK562lysategroups).Ontheotherhand,theinvitroconditionsdeveloped

bytheauthorsofthenewmoleculeshowanintenselabelingbutalsounspecific,asrevealedbythe

smeared lanes (Figure 3-4, initial CuAAC conditions on both recombinant Src-GST and K562 lysate

groups). Thenewly establishedparameters for theCuAAC reactionwith thenew copper-chelating

azidepresentedoptimalresults,withaclearSrc-GSTbandintheassaylaneandclearcompetitionin

the adjacent lane (Figure 3-4, optimized CuAAC in the Src-GST group) aswell as in the full lysate

labeling(Figure3-4,optimizedCuAACintheK562lysategroup).

The newly established parameters for the CuAAC reaction with 20 µM TAMRA copper-

chelating-azide(CC8),100µMCuSO4and2.5mMsodiumascorbateatpH8.5wereusedforfuture

labeling experiments. Further adjustments in concentrations of the corresponding biotin copper-

chelatingazide(CC7)werefine-tunedforcaptureexperiments.


60

3.2.2. CapturingdasatinibtargetsinK562celllysateusingCuAACProteins from100μgofK562cell lysatewereenrichedusingaclickableandphotoreactive

dasatinibcapturecompound(CC6)producedintheYaolaboratoryandtheirinitialresultspublished

in Shiet al. 92. The probeswere clicked to the newbiotin copper-chelating azide (CC 8) using theconditions described in the SpecificMethods (3.3.3Capturing dasatinib targets in K562 cell lysateusingCuAAC,onpage79).

Capture experiments were performed following two different workflows: click and pre-

clicked.

3.2.2.1. Pre-clickedcompoundworkflow

In the pre-clicked workflow, competition samples were pre-incubated with free dasatinib

competitor and assay samples with equivalent volume of DMSO. The complete dasatinib capture

compoundcontainingthesortingfunction(CC9)wasproducedaprioriandaddedtoallsamples.The

sampleswerethenphoto-crosslinkedandenrichedwithstreptavidin-coatedmagneticbeads.

Followingtheenrichment,trypsinwasaddedtothesamplesandtheresultingpeptideswere

recoveredandanalyzedbyLC-MS/MS.

Table3–1showsthesummaryofidentificationsonbothsetsobtainedfromtriplicatesofeach

typeofsample.

Pre-clicked Click

Alignment-

based

MS-based Alignment-

based

MS-based


Student’st-test<0.05(%oftotal)

158

(9.0%)

136

(10.0%)

246

(13.9%)

198

(12.5%)

Assay/Competitionfold-change>2(%oftotal)

69

(3.9%)

50

(3.7%)

186

(10.5%)

155

(9.8%)


7

(0.4%)

7

(0.5%)

48

(2.7%)

47

(3.0%)

Totalkinasesidentified(%oftotalproteins)

93

(5.3%)

77

(5.7%)

93

(5.3%)

78

(4.9%)

Student’st-test<0.05(%oftotalkinases)

14

(15.1%)

14

(18.2%)

17

(18.3%)

12

(15.4%)

Assay/Competitionfold-change>2(%oftotalkinases)

15

(16.1%)

14

(18.2%)

15

(16.1%)

11

(14.1%)

Competedandsignificantkinases(%oftotalkinases)(%oftotalcompetedandsignificantproteins)

6

(6.5%)

(85.7%)

6

(7.8%)

(85.7%)

5

(5.4%)

(10.4%)

5

(6.4%)

(10.6%)

Table3–1:SummaryofidentificationsfromdasatinibcaptureexperimentsinK562lysate.ListofproteinswasobtainedusingMaxQuantv1.3.0.3.Numbers for identificationsbasedonspectralcounts (MS-based)andalignment-basedaregiven.


61

As mentioned previously, dasatinib inhibits the BCR-ABL fusion protein but other proteins,

mainlykinases,havebeendescribedtobetargetedbythissmallmolecule19,117,118

.Infact,inthepre-

clickedworkflowbothBCRandABL1areclearly identifiedassignificantlycompetedproteins inthe

K562lysate,alongsidefiveotherkinases(ABL2,SRC,YES1,RIPK2andEPHB4).Table3–2andFigure

3-5compilethedataforthissetofproteins.

Protein TotalMScounts Enrichment(Assay/DMSO) Specificity(Assay/Competition)Genename

DMSO Assay Comp.Intensityfold-

change(log2)

Student’st-test

(pvalue)Intensityfold-

change(log2)

Student’st-test

(pvalue)

ABL1k 2 55 4 5.4 0.021 3.8 0.022

ABL2k 0 16 0 6.7 0.009 4.0 0.011

BCR 0 48 6 5.8 0.007 3.0 0.008

EPHB4k 0 12 0 4.0 0.036 7.6 0.035

RIPK2k 0 5 0 10.5 0.017 9.4 0.017

SRCk 0 9 0 12.1 0.036 11.1 0.047

YES1k 0 19 5 8.0 0.038 3.5 0.044

Table3–2:ListofcompetedandsignificanthitsindasatinibcaptureexperimentsinK562lysateusingapre-clickedworkflow.TotalMS counts from technical replicates (n = 3) of DMSO control, assay and competition (comp.) samples. Intensity fold-change andsignificancelevelsaregivenforenrichment(assayversusDMSOcontrol)andspecificity(assayversuscompetition)ofhits.k=kinase

Figure3-5:CCMSchartofsignificantlycompetedhitsinDasatinibcaptureexperimentinK562lysateusingapre-clickedworkflow.Boxes: minimum and maximum intensity values for DMSO control (D), assay (A) and competition (C) samples. Black bar: Average ofintensity (n=3) ± SEM. Green triangle: log2 fold-change between average intensities from assay and competition samples. Proteins aredisplayedbyincreasingspecificitypvaluefromlefttoright.

AsseenintheCCMSchart(Figure3-5,above),allhitswereidentifiedinlowabundanceinthe

DMSO control samples and significantly enriched in the assay samples. BCR and ABL1, have been

extensively described as strong dasatinib binders19,108,109,113,114

andwere here identifiedwith high

intensitiesintheassaysrelativelytomostoftheotheralsosignificantlycompetedproteins.YES1was

identifiedwithhighintensityintheassaysamplesaswell;itbelongstothetyrosineproteinkinaseas

ABL1andtotheSRCproteinkinasessubfamily.


62

SRCisalsoidentifiedasasignificantlycompetedproteininthisset.ABL2istheothertyrosine

proteinkinasefromtheABLfamilywithanover-lappingroleinsomebiologicalprocessescarriedout

byABL194,95

andwasalso identifiedasa significanthit fordasatinibusing thismethod.EPHB4 is a

receptor tyrosine kinase previously reported to be inhibited by dasatinib as well113. Lastly, the

serine/threonineproteinkinaseRIPK2 isalsospecificallycaptured in thisworkflow,whichwasalso

previouslydescribedasbeinginhibitedbydasatinib117.

When the targets are surveyed for their interactions using the STRING database119, an

intricatenetworkofbindingandactivitiescanbeobservedbetweenthem(Figure3-6,below).

Figure3-6:STRINGproteininteractionanalysisofsignificantlycompetedproteinsindasatinibcaptureexperimentinK562lysateusingthepre-clickedworkflow.Blue:binding;black:reaction;pink:post-translationalmodification;purple:catalysis;gray:associationincurateddatabases.

With this experiment we have shown that bona fide dasatinib targets can be identified inwholecelllysatesfromK562cellsusingaclickablephoto-reactivedasatinibprobethatwasreactedapriori with a new biotin copper-chelating azide, by a bio-orthogonal chemical ligationmethod, to

enrichandidentifytheinteractionpartnersviaaLC-MS/MSshotgunanalysis.Interestingly,Shietal.92wereunabletoidentifythesetargetsintheirexperimentswithK562lysates,allowingforapossible

interpretationthattheprobedidnothavethesameaffinityasfreedasatinibbutwecannowassure

thatknowndasatinibtargetscanbeaddressedwiththedasatinib-alkynecapturecompound(CC6).

Withtheseresults,wesetouttoevaluatetheproteinsidentifiedwiththeclickabledasatinib-

alkyne capture compound in a K562 lysate but performing the CuAAC reaction after the capture

compoundisphotocross-linkedtothebindingpartner.


63

3.2.2.2. Clickworkflow

In theclickworkflow, thedasatinib capturecompound (CC6wasadded to the lysate in the

assay sample as well as the competition sample that was previously incubated with excess free

dasatinib (CC14).Afterequilibration, the sampleswere irradiatedwithUV light topromotecross-

linking of the probe to the interacting proteins. Next, the counter-probe containing the sorting

function(CC7)andthereagentsfortheCuAACreactionwereaddedand,lastly,thelabeledproteins

wereenrichedwithstreptavidin-coatedmagneticbeads.

Followingtheenrichment,trypsinwasaddedtothesamplesandtheresultingpeptideswere

recoveredandanalyzedby LC-MS/MS.Adetailedmethoddescription canbe found in theSpecific

Methodssection(3.3.3CapturingdasatinibtargetsinK562celllysateusingCuAAC,onpage79).A total of 48 of potential hitswere identified. Fold-changes and correspondingp values for

intensitydifferencesaresummarizedinTable3–3,below.

Protein Enrichment(Assay/DMSO) Specificity(Assay/Competition)Genename Intensityfold-

change(log2)

Student’st-test


change(log2)

Student’st-test

(pvalue)ADH5 10.7 0.007 1.8 0.006

AMMECR1;AMMECR1L 8.1 0.011 2.2 0.044

ANXA6 7.4 0.008 6.5 0.008

ATIC 12.2 0.002 1.4 0.001

BRIX1 0.8 0.096 1.2 <0.001

CALML5 1.8 0.071 1.1 0.032

CBR1;CBR3 7.8 0.019 2.3 0.033

CLIC1 6.6 0.004 1.1 0.010

DBT -1.6 0.004 3.9 0.044

DDX42 9.5 0.013 1.1 0.040

DLST -4.0 0.002 2.5 0.007

ENO1 4.3 0.022 1.4 0.038

EPB41L2 9.6 0.011 1.0 0.031

ETFA 8.1 0.026 1.4 0.047

FTSJ3 0.9 0.013 1.2 0.006

GSTP1 8.0 0.014 1.5 0.022

HARS 4.8 <0.001 1.1 0.001

HIST1H1B 2.9 0.007 1.1 0.023

HIST1H1C 3.1 0.028 1.4 0.045

LCP1 6.0 <0.001 1.9 <0.001

MAPK14k 10.0 0.010 1.7 0.010

MDH2 4.9 <0.001 1.2 <0.001

MMTAG2 -1.4 0.027 6.7 0.012

MYH9 6.2 0.014 1.4 0.020

MYL12B;MYL12A;MYL9 6.1 0.007 1.1 0.019

NANS 10.7 0.005 1.2 0.004

OGDH -5.4 <0.001 1.3 0.001

PFAS 9.5 0.040 2.6 0.036

PFN1 6.3 0.002 1.1 0.002

PGK1k 4.1 0.001 1.1 0.001

PMPCA 10.6 0.007 1.5 0.006


64

Protein Enrichment(Assay/DMSO) Specificity(Assay/Competition)Genename Intensityfold-

change(log2)

Student’st-test


change(log2)

Student’st-test

(pvalue)PREP 4.1 0.001 1.2 0.011

PRPF4Bk 2.1 0.006 1.3 0.022

PSAT1 6.9 0.003 3.2 0.003

TAOK3k 7.8 0.007 1.5 0.046

TOMM5 10.6 0.010 1.4 0.021

TPI1 3.4 0.001 1.5 0.002

TPM3 2.2 0.012 1.2 0.026

TSFM 10.0 0.007 1.2 0.047

TXLNG 9.0 0.001 1.8 0.046

TXNRD1 8.4 0.009 1.1 0.014

U2SURP 9.2 0.027 1.8 0.040

UBA1 11.4 0.015 1.0 0.037

UBA6 8.8 0.006 1.1 0.006

UBE2L3 5.2 0.002 1.4 0.002

UQCRFS1 1.8 0.079 1.5 0.046

UQCRQ 4.5 0.006 1.1 0.013

VRK1k 9.7 0.009 4.5 0.007

Table3–3:ListofcompetedandsignificanthitsinDasatinibcaptureexperimentsinK562lysateusingaclickworkflow.Intensity fold-change and significance levels are given for enrichment (assay versus DMSO control) and specificity (assay versuscompetition)ofhits.k=kinase

Somekinasesare identifiedusingtheclickworkflowin lysate:PGK1,VRK1,MAPK14,PRPF4B

andTAOK3.NointeractionsbetweenthemareknownaccordingtotheSTRINGdatabase(Figure3-7).

Figure3-7:STRINGproteininteractionanalysisofsignificantlycompetedkinasesinDasatinibcaptureexperimentinK562lysateusingtheclickworkflow.


65

According to a recent publication by Kitagawa et al. 118, where tyrosine kinase inhibitors,includingdasatinib,wereprofiledagainstapanelof310kinases,MAPK14showsanIC50of330nM

for dasatinib whereas the small molecule does not inhibit PGK1. TAOK3 was not included in this

studybuttheauthorsprofiledTAOK2,whichwasalsonot inhibitedbydasatinib. Inthestudyfrom

Davis et al. 108, some additional kinases were profiled against dasatinib and results showed that

TAOK3 presented a Kd of 2300µM for dasatinib, VRK2 (not VRK1) a Kd of 3200µM and PRPF4B

showednodata. TheCCMSdata for these significantly identifiedkinases in the clickworkfloware

presentedinFigure3-8.

Figure3-8:CCMSchartofsignificantlycompetedkinasesinDasatinibcaptureinK562lysateusingCuAAC.Boxes: minimum and maximum intensity values for DMSO control (D), assay (A) and competition (C) samples. Black bar: Average ofintensity (n=3) ± SEM. Green triangle: log2 fold-change between average intensities from assay and competition samples. Proteins aredisplayedbyincreasingspecificitypvaluefromlefttoright.

BCR,ABLandSRCwerenotidentifiedassignificantlycompetedintheclickworkflow.Table3–

4presentsthevaluesfortheexpectedtargetsofdasatinibaspreviouslyobservedinthepre-clicked

workflow.

Protein TotalMScounts Enrichment(Assay/DMSO) Specificity(Assay/Competition)Genename

DMSO Assay Comp.Intensityfold-

change(log2)

Student’st-test


change(log2)

Student’st-test

(pvalue)ABL1k 2 10 3 2.1 0.003 0.4 0.018

ABL2k 0 0 0 2.2 0.009 0.2 0.531

BCR 0 6 7 3.3 0.016 0.3 0.262

SRCk 0 0 0 7.3 0.068 6.4 0.069

Table3–4:ValuesforexpectedtargetsofDasatinibinK562lysateusingaclickworkflow.TotalMS counts from technical replicates (n = 3) of DMSO control, assay and competition (comp.) samples. Intensity fold-change andsignificancelevelsaregivenforenrichment(assayversusDMSOcontrol)andspecificity(assayversuscompetition)ofhits.k=kinase


66

Allproteinspresented inTable3–4,onpage65,areenrichedwhencompared to theDMSO

controls. Even in theabsenceof spectral counts,MaxQuant software is able to retrievedata from

otherrunsandsearchfortheintensityofthateventinthesamplebeinganalyzed120.Thisexplains

thepositiveintensityvaluewhennoMScountforaspecificproteinisobservedinthatsample.

Thespecificityfold-changeobtainedforBCRandABL2arepurelyrandom,assuggestedbythe

highpvaluesof0.262and0.531,respectively.However,thelowintensityfold-changebetweenassayandcompetitionforABL1isvalid(p=0.018),suggestingthattheclickabledasatinibprobeaddressesthis target but the overall capture yields are smaller compared to the pre-clicked compound

workflow.Infact,whenlookingatthespectralcountsforthisprotein,theassayclearlyshowsmore

identifiedpeptides(10)thantheDMSO(0)andcompetition(3)controls. Inanoppositesituationis

SRC,wherenoMScountswereidentifiedbutalignmentdatarevealshighenrichmentandspecificity

(log2fold-changeof7.3and6.4,respectively),andeventhoughitdoesnotmeettheinclusioncriteria

ofp<0.05,thedifferencetothethresholdislessthan0.02.When the 48 specific targets obtained in the click workflow are submitted to the STRING

protein interactiondatabase, the loosenetworkdepicted inFigure3-9 (onpage67) isobtained. In

fact, by analyzing further this network searching for enrichment of Gene Ontology terms, “small

molecule metabolic process” and “cellular nitrogen compound metabolic process” are obtained

(Table3–5),suggestingthehitsshownonTable3–3,onpage64,arenotspecifictargetsofdasatinib

butgeneraldrugmetabolizingenzymes.

GeneOntologyTerm–BiologicalProcess NumberOfGenes p-value p-value_FDR p-value_bonferroni

cofactormetabolicprocess 6 3.86E-06 2.61E-02 4.87E-02

cellularaminoacidmetabolicprocess 7 4.14E-06 2.61E-02 5.22E-02

smallmoleculemetabolicprocess 13 3.45E-04 1.00E+00 1.00E+00

cellular nitrogen compound metabolicprocess 18 1.84E-03 1.00E+00 1.00E+00

Table3–5:GeneOntology termenrichment fromsignificantlycompetedproteins inDasatinibcaptureexperiment inK562 lysateusingaclickworkflow.


67

Figure3-9:STRINGproteininteractionanalysisofsignificantlycompetedproteinsindasatinibcaptureexperimentinK562lysateusingtheclickworkflow.Blue:binding;gray:associationincurateddatabases.

Theresultsobtainedusingthedasatinib-alkynecapturecompoundinapost-irradiationclick

usingK562 lysatedonot reflect thepreviouslyobtainedknowntargetsofdasatinib.However, this

probewasdevelopedtobecellpermeableandusedinalivingcellsystem.Therefore,wemovedon

establishing the click capture conditions in living K562 cells knowing the probe is able to identify

bonafidedasatinibtargets,asseenonthepre-clickworkflow.


68

3.2.3. CellviabilityassayfordasatinibcompoundsinK562cellsBeforeperformingcaptureexperimentsinlivingcells,itisimportanttodetermineifdasatinib

and the clickable dasatinib probe affect cell viability at the used concentrations in capture

experimentssinceitcouldintroduceabiasinthesubsequentdataanalysisduetodifferencesincell

numbersandconsequentproteinamounts.

To determine the cell viability of K562 cells after incubation with dasatinib and dasatinib

clickable probe, the CellTiter-Glo® Luminescent Cell Viability Assay from Promega™ was used.

Generally,thismethoddeterminesthenumberofviablecellsinculturebasedonquantitationofthe

ATPpresent,whichsignalsthepresenceofmetabolicallyactivecells.Thekitincludesathermo-stable

luciferasethatprovidestheluminescentsignalthatcanbemeasured.

The experimentwas performed on a 96-well plate,where a dilution series of K562 cells (in

triplicate)wasused toestablisha calibration curve for the correspondingATPpresent ineach cell

dilution. A regression curvewas determined based on the average of themeasured signal. Assay

wellswerepreparedcontaining100.000cellsandeitherdasatinib (CC14–Dasa inFigure3-10)or

clickable dasatinib-alkyne capture compound (CC 6 – CPT392 in Figure 3-10) at different

concentrations and incubation times. The luminescent signals measured in the assay wells were

convertedtoATPconcentrationvaluesandcorrespondingviablecellnumbersbyinterpolationfrom

thecalibrationcurve.Thepercentageofviablecellswasthencalculatedbasedontheinitialinputof

cells.Additionalwellscontainingonlycompoundswithoutcellswerealsopreparedtoruleoutany

effect of the compound on the luminescent signal. Figure 3-10 illustrates the multi-well plate

scheme.

Figure3-10:Schemeofmulti-wellplateforcellviabilityassayofdasatinib(Dasa)anddasatinibclickablecompound(CPT392)inK562cells.WellsA1toC12:DilutionseriesofK562cells.WellsD1toG8:100.000cellswereincubatedfor30or90minwitheitherdasatinib(Dasa)ordasatinibclickablecompound(CPT392)at100nM,500nM,2.5μMand12.5μM.WellsD10toG11:onlycompoundwasadded.

Luminescence signal was obtained using an Imaging System (Syngene G:BOX XT4) and the

imageobtainedwasanalyzedwithImageJsoftwaretoextracttheintensitiesofeachwell.Datawas

furtherprocessedinMicrosoftExceltocalculatetheeffectofthecompoundsontheviabilityofK562

cells(Figure3-11,onpage69).

1 2 3 4 5 6 7 8 9 10 11 12

A 100.000 50.000 25.000 12.500 6.250 3.125 1.563 781 391 195 98 0

B 100.000 50.000 25.000 12.500 6.250 3.125 1.563 781 391 195 98 0

C 100.000 50.000 25.000 12.500 6.250 3.125 1.563 781 391 195 98 0

D100nMDasa

500nMDasa

2.5μMDasa

12.5μMDasa

100nMDasa

500nMDasa

2.5μMDasa

12.5μMDasa

100nMDasa

100nMCPT392

E100nMDasa

500nMDasa

2.5μMDasa

12.5μMDasa

100nMDasa

500nMDasa

2.5μMDasa

12.5μMDasa

500nMDasa

500nMCPT392

F100nMCPT392

500nMCPT392

2.5μMCPT392

12.5μMCPT392

100nMCPT392

500nMCPT392

2.5μMCPT392

12.5μMCPT392

2.5μMDasa

2.5μMCPT392

G100nMCPT392

500nMCPT392

2.5μMCPT392

12.5μMCPT392

100nMCPT392

500nMCPT392

2.5μMCPT392

12.5μMCPT392

12.5μMDasa

12.5μMCPT392

H

#cells(calibrationcurve)

[email protected]/mL

30min1h30 nocells


69

A B

Figure3-11:Luminescencedetectionofcellviabilityassayofdasatinibandclickabledasatinib-alkynecapturecompoundinK562cells.(PanelA)WellsA1toC12:DilutionseriesofK562cells.WellsD1toG8:100.000cellswereincubatedfor30or90minwitheitherdasatinibor clickable dasatinib capture compound compoundat 100nM, 500nM, 2.5 μMand12.5μM.Wells D10 toG11: only compoundwasadded.(PanelB)Plotofnumberofcellsversusluminescenceintensity(rawvolume)fromnon-incubatedcells.Linearregressionofpointsispresented.

Calculated values for the viability of K562 cells after incubationwith dasatinib or dasatinib-

clickablecompoundarepresentedinTable3–6.

%ofviableK562cells

dasatinib dasatinib-alkynecapturecompound

Compoundconcentration 30min 90min 30min 90min

12.5μM 75% 100% 74% 97%

2.5μM 90% 99% 68% 95%

500nM 95% 98% 90% 95%

100nM 99% 93% 95% 93%

Table3–6:Effectofdasatiniborclickabledasatinib-alkynecapturecompoundonK562cellviability.

AsobservedinTable3–6,increasingconcentrationsofbothcompoundsreducecellviabilityto

74-75%whenincubatedforashortperiodof30min.At90minincubations,thiseffectisnolonger

visibleandcellviabilityremainsover90%.Thiscouldindicateacellularresponsemechanismtoboth

small molecules that leads to the increase of ATP production after the initial toxic effect. Also

interesting to notice is that both compounds have the same effect pattern indicating a similar

mechanismofaction.Atlowerconcentrations(100nMor500nM),bothdasatinibandthedasatinib-

alkynecapturecompounddonotappeartobetoxictothecellsdespitetheincubationtime.

Thelackofcytotoxicityisrathersurprisingbutthisabsenteffectcouldbeduetoanacquired

resistance to dasatinib from the particular strain of K562 cells used for the experiments or,more

likely,duetotheshortincubationtimesthatareinsufficienttotriggercelldeath.

Nevertheless, the identificationof dasatinibbinders in living cells by clickCCMSwas carried

outsincethetargetswereconfirmedinthelysateofthesamestrainofcells.

Living cell capture of dasatinib targets was then performed on K562 cells using low

concentrationsofcompoundsandshortincubationtimes.


70

3.2.4. CapturinginlivingK562cellsCurrentsmall-moleculetarget interactionprofiling ismainlydoneinsolution.Certainprotein

conformations are modified during the preparation of lysates and, consequently, putatively

importantprotein-protein interactions are lost.Devising anew strategy to address small-molecule

targets in their natural cellular environments allows for a more accurate profiling of targets and

partner interactions as well as directly demonstrating target engagement. Using a small clickable

compoundallowstoovercomethelimitationsofacompleteprobeintermsofcellularpermeability

andtoreducenon-specificscaffoldinteractions.ACCMSworkflowstrategyforcaptureofdasatinib

targets in living K562 cells was developed using the click conditions previously established and

optimizedinlysates.

Briefly,thecellsaregrowninmulti-wellplatestoafixedconcentrationandsupplementedwith

mediumcontainingdasatinib for the competition samplesorDMSO for the assays.After that, the

clickabledasatinibprobeisaddedtoallwellsandfurtherincubatedinacontrolledatmosphere.The

mediumisremovedandthecellsarewashedwithPBS.TheplatesarethenirradiatedwithUVlight

topromotethecrosslinkingofthereactivityfunctioninthecapturecompoundtotheboundprotein.

Thecellsarerecovered,lysedandthenclickedtothecounter-probecontainingthesortingfunction

using the reagents for the CuAAC reaction and, lastly, the labeled proteins are enriched with

Streptavidin-coatedmagneticbeads.Followingtheenrichment,trypsinisaddedtothesamplesand

theresultingpeptidesarerecoveredandanalyzedbyLC-MS/MS.

A totalofnine individualexperimentseachwith triplicatesampleswereanalyzed.Table3–7

summarizestheresultsobtained.

Experiment A B C D E F G H I

Totalproteinsidentified 1322 1322 884 1096 1430 1653 921 921 1428

Student’st-test<0.05 99 33 3 33 73 30 21 70 197

(%oftotal) (7.5%) (2.5%) (0.3%) (3%) (5.1%) (1.8%) (2.3%) (7.6%) (13.8%)

Assay/Competitionfold-change>2 577 118 16 89 73 65 83 40 160

(%oftotal) (43.6%) (8.9%) (1.8%) (8.1%) (5.1%) (3.9%) (9%) (4.3%) (11.2%)

Competedandsignificantproteins 93 5 0 14 5 4 9 2 14

(%oftotal) (7%) (0.4%) (0%) (1.3%) (0.3%) (0.2%) (1%) (0.2%) (1%)

Totalkinasesidentified 55 55 31 50 69 89 30 30 64

(%oftotalproteins) (4.2%) (4.2%) (3.5%) (4.6%) (4.8%) (5.4%) (3.3%) (3.3%) (4.5%)

Student’st-test<0.05 4 1 0 2 2 3 0 4 16

(%oftotalkinases) (7.3%) (1.8%) (0%) (4%) (2.9%) (3.4%) (0%) (13.3%) (25%)

Assay/Competitionfold-change>2 31 8 1 3 9 7 4 2 6

(%oftotalkinases) (56.4%) (14.5%) (3.2%) (6%) (13%) (7.9%) (13.3%) (6.7%) (9.4%)

Competedandsignificantkinases 3 1 0 1 1 1 0 0 1

(%oftotalkinases) (5.5%) (1.8%) (0%) (2%) (1.4%) (1.1%) (0%) (0%) (1.6%)

(%oftotalcompetedandsignificantproteins) (3.2%) (20%) (0%) (7.1%) (20%) (25%) (0%) (0%) (7.1%)

Table3–7:SummaryofidentificationsfromdasatinibcaptureexperimentsinK562livingcellsinnineseparateexperiments.(A–I)eachconsistingofatleastthreetechnicalreplicates.ListofproteinswasobtainedusingMaxQuantv1.3.0.3.


71

As seen in Table 3–7, on page 70, the number of significantly competed proteins varies

between0and93,withacalculatedmedianof5.Theidentifiedproteinswithinthesenumbersare

alsovariablebetweeneachexperiment,whichhindersthecorrectidentificationofpotentialtargets

fordasatinibusingthisworkflow.

One possible way to extract significant information from this combined set of data is to

develop a scoringmethod to rank the proteins according to the frequency theywere significantly

competed in each experiment. A value of 1 is attributed to each protein that was significantly

competedintheexperimentandthefinalscoreisthesumacrossallexperiments.Usingthisanalysis

method,fourproteinshaveascorehigherthan1,meaningtheyweresignificantlycompetedinmore

thanoneexperiment.Table3–8,onpage72,summarizesthevaluesforspecificityfold-changeand

correspondingpvalueforthesetargetsineachexperiment.Additionally,resultsforABLandBCRare

alsopresentedinTable3–8.SRCwasnotidentifiedinanyexperimentusingthisworkflow.

TheproteinwiththehighestscorewasCSK,anon-receptortyrosine-proteinkinase.CSKwas

identified as specifically competed in 3 out of 9 experiments and is then the highest potential

candidate to be inhibited by dasatinib in a natural cell environment. CSK phosphorylates tyrosine

residues located in theC-terminal tails of Src-family kinases regulating their activity and therefore

plays an important role in the regulation of cell growth, differentiation, migration and immune

response121. It. According to Kitagawa et al. 118, dasatinib has an IC50 of 3.2 nM against CSK.

Additionally, dasatinib binding to CSK in living cells was previously observed in Kim et al. usingmagneticbiotinparticles

122.

AnotherpotentialtargetfordasatinibwasWARS,Tryptophan--tRNAligasethatregulatesERK,

Akt,andeNOSactivationpathwaysthatareassociatedwithangiogenesis,cytoskeletalreorganization

andshearstress-responsivegeneexpression123,124

.Interestingly,anassociationbetweenWARSand

dasatinib was previously reported in a PhD thesis by BrendanMartin Corkery, entitled “Tyrosine

KinaseInhibitorsinTripleNegativeBreastCancer”125.Inthiswork,itwasobservedahighlyconfident

increase ofWARS (annotated as IPF53 in themanuscript) in dasatinib-treated versus non-treated

cellsusingaDIGE-MALDI-TOF/TOFanalysisapproach.

TheothertwopotentialcandidatesfordasatinibtargetswerePSMC4(26Sproteaseregulatory

subunit 6B) andRPS17 (40S ribosomal protein S17).No literature about any interactions between

theseproteinsanddasatinibwasfound.

Insummary,theprofilediffersfromwhatisobservedinthelysate.Thiscouldbeattributedto

a lower efficiency of the method or that living cells are a more complex system, though more

realistic,makingtheanalysismoredifficult.


72

Specificity(Assay/Competition) Intensityfold-change(log2) Student’st-test(pvalue)

ExperimentA B C D E F G H I A B C D E F G H I

Genename Score

CSK 3.87 9.19 #N/I 0.09 1.02 0.63 #N/I #N/I 3.86 0.210 0.005 #N/I 0.950 0.040 0.685 #N/I #N/I 0.001 3

WARS 1.86 6.21 -1.08 1.18 0.23 0.16 0.65 -0.18 0.64 0.031 0.425 0.480 0.048 0.633 0.645 0.196 0.599 0.012 2

PSMC4 2.59 0.69 0.13 0.68 -0.23 -1.02 -0.01 0.31 1.24 0.014 0.287 0.786 0.241 0.261 0.572 0.973 0.073 0.042 2

RPS17;RPS17L 4.60 0.54 #N/I 0.75 -0.61 -1.26 0.32 0.20 1.06 <0.001 0.461 #N/I 0.471 0.209 0.608 0.545 0.279 0.020 2

ABL1;ABL2 6.42 0.24 #N/I -0.76 0.05 #N/I -0.21 -0.09 1.34 0.022 0.797 #N/I 0.405 0.685 #N/I 0.099 0.926 0.156 1

BCR 3.18 -0.13 -0.89 0.50 0.43 #N/I #N/I #N/I 0.68 0.003 0.883 0.482 0.591 0.356 #N/I #N/I #N/I 0.351 1Table3–8:ExcerptofpotentialhitsfordasatinibinK562livingcellcaptureusingaclickworkflowfrom9individualexperiments(A–I)eachconsistingoftriplicatesforassaysandcompetitions.Valuesinboldmarkspecific(log2fold-change>1)andsignificant(pvalue<0.05)resultsforagivenexperiment.Scoreiscalculatedbythesumofnumberofresultsthatfulfilledboththecriteriaofspecificityandsignificance.#N/I:notidentified


73

Analternativeanalysismethodtobypassthevariabilityobservedbetweeneachexperimentisto analyze all samples together as technical replicates, calculating a unique fold-change andassociated p value between the average normalized intensity obtained from all assays andcompetitions.

With this method of analysis only two proteins are identified as significantly competed bydasatinib:ALDOC(Fructose-bisphosphatealdolaseC)and,onceagain,CSK.

Table3–9liststhefold-changeandpvaluesforthetop20competedproteins(approximatelytop1%ofallidentifications)whenthesearesortedbyincreasingstatisticalsignificance.

Protein Specificity(Assay/Competition) Protein Specificity(Assay/Competition)





ALDOC* 0.97 0.008 NHP2 4.48 0.112CSK*,k 1.33 0.044 AZGP1 1.72 0.121HBA1 2.19 0.050 SARS 0.55 0.122GSTO1 0.85 0.073 NAP1L1 1.19 0.123ESF1 1.92 0.084 RAB21 1.20 0.125FAU 7.55 0.087 GAR1 1.78 0.134ABL2;ABL1k 3.33 0.088 BCR 0.75 0.136KRI1 5.27 0.099 AHCY 0.45 0.139TSR1 2.86 0.104 GARS 0.54 0.140EEF2 0.63 0.112 SLC1A5 0.36 0.145

Table3–9:Top20list(sortedbyincreasingpvalue)ofdasatinib-competedproteinswhencalculatingvaluesforallrunsfromallexperiments

26assaysand26competitions.*=significantandcompeted;k=kinase

The results obtained for ALDOC, CSK, ABL and BCR in each individual experiment can be

observed in Figure 3-12, on page 74. ALDOCwas the protein from this listwhichwasmore oftenidentified across experiments but reduced intensity values in the competitionwere only observedsignificantly in two experiments (A and F) as opposed to CSK which, as said previously, wassignificantlycompeted in threeseparateexperiments (A,Eand I).BCRandABLareonlycompetedwithasignificantpvalueinoneexperiment(A).

Analyzingtheoverallresults, itbecomesmoreapparentthatdasatinibprimarilyengagesCSKinK562cells.Infact,CSKwasoneofthetoptyrosinekinasesidentifiedbyShietal.whenperformingthe invitroclickcapture inK562 lysate 92.Also,CSKhasbeenpreviouslycapturedwithadasatinibcapture compound in HepG2 lysate using the classical CCMS workflow 13 and in the analysisperformedbyDavisetal.108dasatinibinhibitsCSKwithaKdof1nM(thisproteinisrepresentedinFigure 3-3, on page 56, as being inhibited by dasatinib but not by the other represented tyrosinekinaseinhibitors).


74

Figure3-12:CCMSchartsofCSK,ALDOC,ABLandBCRproteinsinDasatinibcaptureexperimentinK562livingcellsusingaclickworkflow.

Boxes:minimumandmaximumintensityvaluesforassay(A/blue)andcompetition(C/red)samples.Blackbar:Averageofintensity(n=3)

±SEM.Greentriangle:log2fold-changebetweenaverageintensitiesfromassayandcompetitionsamples.*significantfold-change(pvalue

<0.05)


75

When the protein list from Table 3–9, on page 73, is analyzed with STRING, a network ofenriched interactions is obtained (Figure 3-13).Most of the interacting proteins are associated toribosomesortothecellulartranslationalmachinery.Theothersmallernetworkobserved inFigure3-13 isbetweenABL1,ABL2andBCRwhileallother identifiedproteinshave little interactionwitheachotheraccordingtothisdatabase.

Figure3-13:STRINGproteininteractionanalysisofsignificantlycompetedproteinsindasatinibcaptureexperimentinK562lysateusingthe

clickworkflow.

Blue:binding;black:reaction;pink:post-translationalmodification;purple:catalysis;gray:associationincurateddatabases.

Furtheranalysisof thenetwork forGeneOntology terms revealsa significantenrichmentof

protein tyrosine kinases as well as other functions related to general small molecule bindingproperties (Table 3–10). Lastly, correlation analysis of this network for related diseases identifieschronicmyeloidleukemiaasatopcandidate,whichisinaccordanceofdasatinibtherapeuticuseandK562cellbiology.

GeneOntologyTerm–MolecularFunctionNumberOfGenes

pvalue pvalue_FDR pvalue_bonferroni

poly(A)RNAbinding 8 3.94E-06 7.62E-03 1.51E-02

heterocycliccompoundbinding 14 6.71E-06 7.62E-03 2.57E-02non-membrane spanning protein tyrosinekinaseactivity

3 6.75E-06 7.62E-03 2.59E-02

organiccycliccompoundbinding 14 7.94E-06 7.62E-03 3.05E-02Table3–10:GeneOntologytermenrichmentfromsignificantlycompetedproteinsindasatinibcaptureexperimentinK562livingcellsusing

aclickworkflow.


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Althoughthefirstsystemestablishedtoidentifysmallmoleculetargetsinlivingcellsviaabio-orthogonalchemicalligationmethoddidnotdelivertheexpectedresults,importantinformationcanbe retrieved. Living cell systems have inherent high variability in several structural, biological andbiochemicalcharacteristicsduetodifferentcellcyclestages,whichcaninfluencetheuptakeofsmallmoleculesandintroduceunforeseenbias.Onewaytotacklethisissuecouldbetosynchronizecellspriortoexperimentationbutthiswouldbelessrepresentativeofhowdynamicabiologicalsystemisandthemainobjectiveofintroducingthisnewapproachtoidentifysmallmoleculetargetsistobeasclosetothenaturalsystemaspossible.Analternative,whichwasconfirmedinthefinalanalysis,istoincreasethenumberofbiologicalandtechnicalreplicatestohavestrongstatisticsandconfidenceintheobtainedresults.

With this inmind, the next stepwas to identify targets from a smallmolecule with poorlyunderstoodmodeofaction.

Chapter3:IdentifyingDasatinibtargetsincells|SpecificMethods

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3.3. SPECIFICMETHODSFORTHEDASATINIBSYSTEM3.3.1. StructuresofusedcompoundsCC5:ClickableTAMRA[tetramethylrhodamine]azide(containslabelingfunction)

CC6:ClickableDasatinibalkynecapturecompound(containsselectivityandphotoreactivefunctions)

CC7:Clickablebiotin-PEGcopper-chelatingazide(containslabelingandcopperligandfunctions)

CC8:ClickableTAMRA-PEGcopper-chelatingazide(containslabelingandcopperligandfunctions)


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CC9:Clickeddasatinib-biotincapturecompound.ReactionproductofCC6andCC7

CC14:Dasatinibcompetitor

CC18:Copper-chelatingligandBTTEbis(tert-butyltriazoly)ethanol

3.3.2. Optimizing CuAAC reaction parameters with new copper-chelatingazide

160pmolof recombinanthumanSRCor50µgofK562 cell lysatewasdistributed in200µLPCR-tubestripsanddilutedwithwaterand5XCaptureBuffer.Tothecompetitionsamples,500µMofdasatinibcompetitor(CC14)wasaddedandthecorrespondingvolumeofwaterwasaddedtotheassay samples. The vials were incubated for 10 min on ice and then 20 µM of dasatinib-alkynecapturecompound(CC6)wasadded.Thesampleswere incubated for90minrotatingat4 °Candthen irradiated for 10 min at 350 nm in a caproBox™ to promote photo-crosslinking of thecompounds to the binding targets. The CuAAC reaction was performed in three different sets tocompareefficiencies:(i)previouslyestablishedconditionswith20µMTAMRA-azide(CC5),100µMCuSO4, 200 µMBTTE (CC 18) and 2.5mM sodium ascorbate at pH 8.5; (ii) Bevilacqua’s chemistryparameters with 10 µM TAMRA copper-chelating-azide (CC 8), 200 µM CuSO4 and 5mM sodiumascorbate; (iii) newlydesignedmixturewith20µMTAMRAcopper-chelating-azide (CC8), 100µMCuSO4and2.5mMsodiumascorbateatpH8.5.Theclick reaction ran for30minshakingat roomtemperature.ConcentratedLaemmlibufferwasaddedtothesamplesandthenboiledfor10minat100°C.

TheresultswerevisualizedbySDS-PAGEfollowedbyBlottingwithchemiluminescencereadoutonx-rayfilmusingIgGanti-TAMRAfollowedbyanti-IgG-HRPsecondaryantibody.

BandintensitywasmeasuredusingImageJsoftwareforfold-changecalculations.


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3.3.3. CapturingdasatinibtargetsinK562celllysateusingCuAACK562 cell lysate was prepared as described in the General Methods (6.1.2.1 K562 lysate

preparation,onpage116).A full dasatinib-biotin capture compound (CC 9) was prepared a priori to be used as a

reference:500µMofbiotincopper-chelating-azide(CC7)wasequilibratedwith5mMofCuSO4for10minatR.T.andthenreactedwith500µMofdasatinib-alkynecapturecompound(CC6)inPBSatpH8.5byadding2.5mMofsodiumascorbate.Thevialwasshakenvigorouslyfor10minatR.T.

100 µg of K562 cell lysate was distributed in 200 µL PCR-tube strips and either 125 µM ofdasatinibcompetitor(CC14)wasaddedtothecompetitionsamplesorthecorrespondingvolumeofDMSOwasaddedtotheassaysamples.Thevialswereincubatedfor30minoniceandthenadded5µM of dasatinib-alkyne capture compound (CC 6) for post-click samples or 5 µM of pre-clickeddasatinib-biotin capture compound (CC 9) to the pre-click samples. To additional control sampleswithorwithoutdasatinibcompetitor,theequivalentvolumeofwaterwasadded.Allsampleswereincubated for90min rotatingat4 °Cand then irradiated for10minat350nm inacaproBox™topromote photo-crosslinking of the compounds to the binding targets. The CuAAC reaction wasperformedinthesamplescontainingthedasatinib-alkynecapturecompound(CC6)byadding5µMofbiotincopper-chelating-azide(CC7) (pre-equilibratedwithtwo-timesexcessCuSO4)and2.5mMofsodiumascorbate.Tocontrolandpre-clickedsamples,theequivalentvolumeofPBSwasadded.Theclickreactionranfor30minshakingatroomtemperature.

Streptavidin-coatedmagnetic beadswerewashedwith PBS and incubatedwith the samplesfor1h rotatingat4 °C.Thebeadswere recoveredusingacaproMag™and thenwashedsix timeswithwashbufferfollowedbythreetimeswashwith80%acetonitrileandafinalwashwithMS-gradewater.

0.5µgoftrypsininammoniumbicarbonatewasaddedtothebeadsandincubatedover-nightshakingat37°C.Thesupernatantcontainingthetrypticdigestwasrecoveredtocleantubes;next,the beads were washed with MS-grade water and the wash solution pooled with the originalsupernatant. All samples were lyophilized and analyzed by LC-MS/MS following the protocoldescribedintheGeneralMethodssection(6.3MassSpectrometry,onpage121).

3.3.4. CellviabilityassayfordasatinibcompoundsinK562cellsTo determine the cell viability of K562 cells after incubation with dasatinib (CC 14) and

clickable dasatinib-alkyne capture compound (CC 6), the CellTiter-Glo® Luminescent Cell ViabilityAssayfromPromega™wasused.

CellTiter-Glo® Reagent was prepared as described by the manufacturer and the assay wasperformed in triplicate inanopaque96-wellmultiplate.Toestablishacalibrationcurve,100µLofK562cellsataconcentrationof1x106cells/mLinRPMImediumwasdispensedinthestartingwellanddilutedsequentiallyinthesamemediumwithadilutionfactoroftwo.Controlwellscontainingmedium without cells were also prepared to determine the value for background luminescence.Assay wells were prepared with 100.000 cells and either dasatinib (CC 14) or dasatinib-alkynecapturecompound(CC6)wereaddedandincubatedfor30or90minat37°C(5%CO2).Theplate


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anditscontentswasequilibratedatroomtemperaturefor30min.100µLofCellTiterGlo®Reagentwasaddedtothewellsandtheplatewasmixedfor2minonanorbitalshaker.Theplatewasthenallowedtoincubatefor10minatroomtemperature.

The luminescence signal was recorded in an Imaging System (Syngene G:BOX XT4) and theimageobtainedwasanalyzedwiththeImageJsoftwaretoextracttheintensitiesofeachwell.DatawasfurtherprocessedinMicrosoftExceltocalculatetheeffectofthecompoundsontheviabilityofK562byinterpolationfromthecalibrationcurve.

3.3.5. CaptureofdasatinibtargetsinlivingK562cellsusingCuAACK562cellswereressuspendedinRPMI1640mediumwithoutphenolred,supplementedwith

10%FBSand1%penicillin/streptomycin,whenusedforexperiments.1x106K562cellswereplatedonto 6-wells multiplates and left over-night in a cell incubator at 37 °C (5% CO2). The cells werepelleted for 5 min at 1.000 xg, ressuspended in serum-free RPMI medium and returned to theiroriginalwells.

10 µM of dasatinib competitor (CC 14) or equivalent volume of DMSO was added to thecompetition wells or to the assay, respectively. The plates were returned for 15 min to the cellincubator.Then,1µMofdasatinibalkynecapturecompound(CC6)wasaddedtoallwellsandthecellswere further incubated for90minat37 °C (5%CO2).Theplateswereplaced inaCaproBox™and irradiated for10minat 350nm.The cellswere recovered to tubes andpelleted for5minat1.000xg,andwashedwithD-PBS.

The treated cellswere lysed for 30min on icewith 100 µL of RIPA buffer (50mM Tris-HClpH8.0,150mMNaCl,1%IGEPALCA-630,0.5%Sodiumdeoxycholate,0.1%SDS)supplementedwith0.2mMDTT,0.5µL/mLbenzonaseandproteinaseinhibitors.Thecrudelysatewastransferredto500µLeppiesandcentrifugedfor10minat15.000r.p.m.toremovecelldebris.

The supernatant containing the solubilized proteinswas transferred to 200µL PCR strips towhichwasadded1µMbiotincopper-chelatingazide(CC7)(previouslyincubatedwith10-foldexcessCuSO4) and 2.5 mM sodium ascorbate. The click reaction ran for 30 min shaking at roomtemperature.

Streptavidin-coatedmagnetic beadswerewashedwith PBS and incubatedwith the samplesfor1h rotatingat4 °C.Thebeadswere recoveredusingacaproMag™and thenwashedsix timeswithwashbufferfollowedbythreetimeswashwith80%acetonitrileandafinalwashwithMS-gradewater.


Chapter4:Phenobarbital–newbindersforanolddrug|Introduction

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4. Phenobarbital:newbindersforanolddrug4.1. INTRODUCTION

Phenobarbital (PB)was first produced in 1912 by Bayer and commercialized as Luminal®. Itwas widely used as a hypnotic/sedative for the treatment ofepilepsyuntilthelaterintroductionofbenzodiazepines,almost50years later.Tothisday,PBcontinuestobeadministeredtopatients with established status epilepticus, the most severeformofepilepsyusuallyresistanttobenzodiazepinetreatment,despite phenobarbital’s suboptimal safety profile 126. PB isknown to promote hepatocellular carcinomas in both rat andmice livers but, interestingly, the same effect has not beenobserved in humans 127, and the pathway associated to thismechanismofactionremainstobeelucidated.

It is largely known that PB induces the expression ofspecific cytochrome P450 (CYP) genes, amongst othermetabolizing enzymes present in liver microsomes 127–130. TheCYP superfamily of proteins, comprised of at least 27 distinctgenefamilies,isacollectionofstructurallyrelatedhaemoproteinmono-oxygenaseenzymesthathydroxylatea largenumberofsteroidhormones,fattyacids,drugs,carcinogens, and environmental chemicals 129. In rat liver, PB induces mainly the expression ofCYP2B1andCYP2B2butonlywithhighconcentrationsofthedrug.ThePBreceptor,ifittrulyexists,has lowaffinity forPB127.EventhoughCYP2B1andCYP2B2aretheprimarymembersof theP450superfamily inducedbyPB,theydonotappeartometabolizethedrug127.ThisknowninductionofCYPs by PB is actually used as reference to determine the hepatotoxicity of other drugs andcategorizethemas“PB-like”induceriftheresponseispositive130.

There are several reasons to think that “PB-type” induction of CYPs is involved in hepatictumorpromotionbyPB.AdetailedanalysisonPBmechanisticdataand riskassessmenthasbeencovered byWhysner et al. 127 Induction by PB of P450 enzymes is part of a complex, pleiotropicresponse.PBhasmanyeffectsonliverthatmayormaynotbemediatedbyP450induction,suchaseffectsongapjunctions,effectsonapoptosis,effectsonEGFreceptors,andeffectsonproliferation.Infact,thereareparadoxicaleffectsofPBonmousehepatocarcinogenesis131,makingitdifficulttoextrapolateexperimentaldatafromlaboratoryanimalstohumanriskassessment.Inratsandmicepre-initiatedwithgenotoxiccarcinogens,PBadministration increases thenumberofhepatocellulartumors by approximately 5-fold despite its non-genotoxicity. However, in mice PB occasionallyexhibits strong inhibitory effects on hepatocarcinogenesis initiated with the potent carcinogendiethylnitrosamine.Bothpositiveandnegativeeffectsofphenobarbitalonhepatocyticproliferationandapoptosis,whicharemechanistically involved in thepromotionstageofhepatocarcinogenesis,havebeendescribed.

Understanding the activation mechanism through which PB induces CYP expression mightprovecrucialtoabetterclarificationoftheobservedinter-specieseffectvariance.

Figure4-1:Luminal(phenobarbital)asitwas

packagedin1940/1945.

Picture from product on display at the

StadtgeschichtlichesMuseumLeipzig.


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Several studies have demonstrated that intracellular phosphorylation events control the PBinduction of CYP2B genes 132–134, with some suggesting that both energy status and nutritionalenvironmentof the cell can influencePB regulationofCYP2Bgeneexpression, although theexactmechanismwasnotunderstoodat the time. In2005,Rencureletal.providedevidence thatAMP-activatedkinase(AMPK)mediatesthePBinductionofCYP2Bgenesexpression135,whichtheylaterconfirmedwithAMPKknockoutexperiments136.AMPKisaubiquitouslyexpressedmetabolic-stress-sensing protein kinase that regulates metabolism in response to energy demand and supply bydirectlyphosphorylatingrate-limitingenzymesinmetabolicpathwaysaswellascontrollinggeneandproteinexpression.Iftheenergystockdecreases,AMP/ATPratioincreasesfollowedbyactivationofAMPKwhich subsequently turns off ATP-consuming pathways such as fatty acid, triglycerides andcholesterol synthesis as well as protein synthesis and transcription, and switches on catabolicpathways suchas glycolysis and fatty acidoxidation.AMPKactivity is activatedbyawidearrayofmetabolicstresses,includinghypoxia,ischemia,andoxidativeandhyperosmoticstresses137.

In the proposed mechanism by Rencurel et al. 135,136, PB increases the AMP/ATP ratio thatactivatesAMPKthroughphosphorylationofitsThr172,areactionlaterproposedtobecarriedoutbythe LKB1enzyme 138.A connectionbetweenAMPand itsmainbinderPKA (alreadydescribed in apreviousChapter)and thePBeffectonCYPexpressionhadbeenestablishedby Joannardetal. 139who observed a competition between the two systems, suggesting that the activation of theAMP/PKApathwaydown-regulatedtheinductionofCYP2B1/2byPB.

In 1999, Kawamoto et al. 132 discovered that the constitutive androstane receptor (CAR),sometimesalsoreferredtoasconstitutivelyactivereceptor,translocatedfromthecytoplasmtothenucleus upon PB stimulus. CAR was originally described as a regulator of gene expression fromretinoic acid-responsive elements and is now associated to a series of regulation mechanismsinvolvedinthemetabolismandtransportofvariousxenobioticsandendogenouschemicals140.UponPBtreatment, thesignalingcascade leadstoarecruitmentofCAR inthenucleuswhere itbindstothe phenobarbital responsive enhancer module (PBREM) present in the CYP2B gene family andinduces the expression of P450 enzymes. In 2007, Shindo et al. 141, established the missing linkbetween AMPK activation/phosphorylation and CAR nuclear translocation to enhance CYP2Bexpression, confirming thatPButilizes theAMPKpathwayas signalingmolecules leading toCYP2Bgene expression, although not exclusively with data suggesting the presence of other activationpathways.

A more cohesive theory for the phenobarbital activation and signaling pathway has arisenduringthedevelopmentoftheworkherepresented.Mutohandco-workers142discoveredthatPBindirectlyactivatesCARbyinhibitingtheepidermalgrowthfactorreceptor(EGFR).TheactivationofCAR through dephosphorylation is catalyzed by phosphatase PP2A when associated todephosphorylatedRACK1.DephosphorylatedCARtranslocatestothenucleus,interactswithretinoidXreceptor(RXR),andactivatesthetranscriptionofCYP2BthroughthePBREMenhancer.InhibitionofEGFRwithPBprevents the receptor tobeactivatedand impedesRACK1phosphorylation,possiblythroughtheSrckinase,allowingitsassociationwithPP2AandsubsequentCARactivation.

TheinteractionofCARwiththeEGFRsignalingpathwaywaspreviouslydescribedbyMeyeretal. in 1989 143,where the results showed the affinity of EGF to its receptorwasdiminished in PB-treatedratprimaryhepatocytesalthoughtheabundanceofEGFRwasunchanged.Theexperimentsperformed at the time indicated the inhibitionwas not a direct competition for the receptor but


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acted through some other underlying mechanism. Even though earlier results contradicted thecurrent findings, the author of the initial research has published a letter in support of Mutoh’sachievements144.

ArecentpublicationprovidesevidenceforthemissinglinkbetweenMutoh’sproposalandtheobservedAMPKphosphorylationinassociationwithCARtranslocation.Josephetal.145haveshownthatAMPKisdephosphorylatedbyPP2A,thesamephosphataseshowntocoordinatewithRACK1todephosphorylateCARand lead to the translocationof the latter towards thenucleus. PBbinds toEGFR preventing its activation and RACK1 phosphorylation, which binds to PP2A, preventing thephosphatase to continueactingonAMPKand thus leads toan increaseonAMPKphosphorylationlevels.

AschemedepictingtheproposedmechanismisillustratedinFigure4-2.

Figure4-2:PhenobarbitalinducesCYP2BexpressionviaEGFRinhibition

Epidermalgrowthfactor(EGF)bindingtoitshigh-affinityreceptor(EGFR)inducesdimerizationandautophosphorylationofitsintracellular

tyrosines.Theactivatedreceptorgeneratesadaptingproteindockingsitestotriggerseveraldownstreameffectorpathways,includingthose

stemmingfromthetyrosine-specificphosphorylationofSrc.ActivatedSrcinturnphosphorylatesRACK1,thusinactivatingRACK1-assisted

dephosphorylationofCAR. IncreasingAMP:ATP ratiosactivates cytosolicAMPK, viaphosphorylationby LKB1, increasingATP-generating

intracellular processes such as glycolysis and fatty-acid oxidation. TheAMPK activity is regulated by PP2A,which dephosphorylates the

kinase. Phenobarbital (PB) binding to EGFR prevents the receptor’s activation, suppressing RACK1 phosphorylation and promoting the

binding toPP2A. TheRACK1:PP2A complex recruits CARand catalyzes its dephosphorylation.DephosphorylatedCAR translocates to the

nucleus, interacts with the retinoid X receptor (RXR), and activates the transcription of CYP2B through the phenobarbital responsive

enhancermodule (PBREM).Adapted fromMeyerand Jirtle144, basedonMutoh et al.

142 and Josephet al.

145 using ServierMedicalArt

templates.

Chapter4:Phenobarbital–newbindersforanolddrug|Results

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4.2. RESULTS4.2.1. Determiningpermeabilityofphenobarbital-alkynecapturecompounds

Three phenobarbital alkyne capture compounds bearing different photo-reactivity moietieswere synthesized by the Medicinal Chemistry group at caprotec bioanalytics (Figure 4-3). Thepermeability of the compounds was evaluated externally by Pharmacelsus (in Saarbrücken,Germany)usingaPAMPA*assay.Theconcentrationofeachcompoundismeasuredintheacceptoranddonorwellsatthebeginningandendoftheassaytocalculatethepercentageofflux.TheassaywasperformedintriplicateandtheresultsaredetailedinTable4–1.

Figure4-3:Synthesizedphenobarbital-alkynecapturecompounds

%Flux Mean[%]

SDStartingSolution[nM]

Meanrecovery

[%]Classification

Compound Assay1 Assay2 Assay3

A(CC10) 74.1 30.0 53.8 52.7 22.1 7467.1 5.0 Highpermeability

B(CC11) 39.6 52.2 41.5 44.4 6.8 8897.1 57.7 Mediumpermeability

C(CC12) 5.7 8.0 6.2 6.6 1.2 6894.8 6.3 Lowpermeability

Table4–1:PAMPAresultsofphenobarbitalalkynecapturecompounds.

Thepermeability of phenobarbital alkyne capture compoundswas calculatedbymeasuring its concentration in thedonor andacceptor

wells(n=3).Flux(%)=(acceptorwell)/sum(donorwell+acceptorwell)x100x2

CompoundAhasameanfluxof52.7%,whichclassifiesitasbeinghighlypermeable.However,

themeanrecoverywasonly5%,suggestingthatitistrappedintheartificialmembrane.CompoundBhasasmallerpermeability(44.4%)comparedtothefirstcompoundbutthehighermeanrecoveryof57.7%makesitagoodcandidatetobeusedinbiologicalexperiments.Finally,compoundCshowedlittle permeability with a mean flux of 6.6% and a recovery of 6.3%, which suggests that thiscompoundwasdegradedduringtheassay.

AlthoughcompoundBappearedtobethebestcandidatetobeusedinCCMSexperiments,thelowyieldsinitssynthesisdidnotallowforittobeproducedinenoughquantityforallexperiments.Therefore, compound A was selected to be used in the identification of phenobarbital targets inlivingcellsusingaclickablephenobarbitalalkynecapturecompound(CC10).

*PAMPA:ParallelArtificialMembranePermeationAssayisamethodthatdeterminesthepermeabilityofsubstancesfromadonorcompartmentdiffusingthroughalipid-infusedartificialmembraneintoanacceptorcompartment.


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4.2.2. DevelopingaphenotypicassaytoevaluatephenobarbitaleffectSelection of adequate experimental systems is key in understanding the underlying

mechanismsofactionofaspecificdrug.In1984,Ferroetal.146showedtheinductionofcytochromeP450 (CYP) by phenobarbital in a rat hepatoma cell line (MH1C1),making it a good candidate as abiological system to identify phenobarbital targets and characterize its phenotypic effect. A laterstudy on the effect of phenobarbital on some CYPs and other liver metabolizing enzymes in sixdifferentrathepatomacelllinesconfirmedthatMH1C1cellscomparedbettertoprimaryhepatocytesintheirexpressionofbiotransformationactivities147.Thegoalofimplementingaread-outsystemfortheeffectofphenobarbitalistouseitlateroncepotentialtargetcandidatesareidentifiedbyCCMSandmeasureifablatingorreducingtheirexpressionproducesthesametypeofresponseobservedwiththeuseofthesmall-molecule.

To develop a phenotypic assay, three different approaches with known phenobarbitalresponses,asdescribedintheliterature,tophenobarbitalstimulationwerepursuedsimultaneously:constitutiveandrostanereceptor translocation;AMPactivatedproteinkinasephosphorylation;andcytochromeP450induction.

4.2.2.1. ConstitutiveAndrostaneReceptor(CAR)translocation

The constitutive androstane receptor (CAR) is a known responder to phenobarbital stimulus148–150viaanindirectactivationmechanism.PhenobarbitalactivatesapathwaythattranslocatesCARfromthecytoplasmtothecellularnucleuswhereitwillinducegeneexpression132.

MH1C1 cells were submitted to cellular fractionation using a commercially available kit thatisolates nuclei from cytoplasm as described in the specific methods section (4.3.3 Subcellularfractionation ofMH1C1 cells, on page 106). The isolated fractionswere resolved by SDS-PAGE andtransferredontonitrocellulosemembraneforprobingusingaspecificanti-CARantibody(Figure4-4).

Figure 4-4: Western Blot analysis of CAR in

isolatednucleiandcytosolfromMH1C1cells.

Expected molecular weight for CAR: 46 kDa

(arrowindicatespossibleCAR)

The anti-CAR blot shown inFigure 4-4 reveals that the

antibody produces considerable background, having the strongest reaction at unexpected 55 kDa.The expected reaction for CAR at 46 kDa is very faint in the pre-fractionated samples (Figure 4-4,lanes1and2),mostlyabsentinthecytosolfraction(Figure4-4,lane3)butbecomingclearerinthenuclear fraction (Figure 4-4, lane 4). The cells used in this experiment were not treated withphenobarbital therefore thepresenceofCAR in thenucleusand its absence in the cytoplasmwasunexpected,suggestingapre-activationofthepathway.


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AnewexperimentwasdesignedtoevaluateiftheresultpresentedinFigure4-4confirmsthepresenceofCARinthenucleusinuntreatedcellsorifupontreatmentwithphenobarbitalmoreCARisrecruitedtothissubcellularfraction.

MH1C1cellsweretreatedwithphenobarbitalatdifferentconcentrations(0,1µM,10µM,100µM,250µMor500µM)during1h,2h,4hor6h.ThedetectionofCARineachsubcellularfractionbywesternblotisshowninFigure4-5.

Figure4-5:EffectofphenobarbitalonCARtranslocationfromcytosoltonucleusinMH1C1cells.

Cellswereincubatedwith0–500µMphenobarbitalfor1h,2h,4hor6hfollowedbysubcellularfractionation.Theindividualfractions

wereanalyzedbywesternblotforCARdetection.ArrowindicatespossibleCAR.ExpectedmolecularweightforCAR:46kDa

The results presented in Figure 4-5 show once again the high background created by theantibody used for the detection of CAR. The expectedmolecularweight for CAR (46 kDa) can beattributed to several bands detected between the 55 kDa and 35 kDa markers in the cytosolicfractions independently of the different phenobarbital stimulus (Figure 4-5, lanes 1 – 6). Thestrongestreactionobserved inthepicture isabandslightlyoverthe35kDamarker inthenucleusfraction(Figure4-5, lanes7–12)which, if in factcorrespondstoCAR,confirmsthattheprotein isalreadypresentwithoutphenobarbital stimulus (Figure4-5, lane7) and the intensityof thatbanddoesnotshowlinearitywithincreasingphenobarbitalconcentrationsortimeofstimulation.

The overall results are not clear and suggest that CAR translocation is not a good readoutsystemforphenobarbitaleffectonMH1C1cells.


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4.2.2.2. PhosphorylationofAMPactivatedproteinkinase(AMPK)

AMP-activated kinase (AMPK) is describedas a cellular energy sensor, sensitive toAMP:ATPratios,whichisactivateduponphosphorylationofitsthreonine172(T172)residue151.Rencureletal.135,136 have demonstrated a relationship between phenobarbital stimulus and AMPK activation viaT172phosphorylationinhepaticcellsfromdifferentspecies.

MH1C1 cells cultured in complete DMEMmedium (10% FBS, 1% pen/strep) were starved infreshserum-freemediumpriortotheincubationwithphenobarbital.Thecellswereincubatedfor1h,2h,4hor6hwithphenobarbitalat0,1µM,10µM,100µM,250µMor500µM.Aftertreatmentthemediumwas removed, thecellswerewashedwithD-PBSandrecovered for fractionation.ThecytosolicfractionsofeachsamplewereanalyzedbywesternblotusingantibodiesagainsttotalAMPKandspecificphosphorylationonT172(pAMPK).TheresultsarepresentedinFigure4-6,onpage88.

The results of phenobarbital effect on AMPK phosphorylation in MH1C1 cells after serumstarvationshowthatconcentrationsofphenobarbitalhigherthan10µMreducetheoverallamountofAMPKinthecytosol(Figure4-6,lanes13–24,middlepanel),onlyafter1hincubation,whichisalsoobtainedwith10µMphenobarbitalbutwith6h incubation time (Figure4-6, lane12,middlepanel). The phosphorylation of AMPK (pAMPK) fluctuates over time with a tendency to bemaintained at higher concentrations of phenobarbital but still in fewer amounts compared to thesmallertimeincubation(Figure4-6,toppanel).

Looking at the control samples, which were not stimulated with phenobarbital (Figure 4-6,lanes1–4) it is clear thatAMPKactivation/phosphorylation is cyclic.After1hof the startof theexperiment, AMPK is phosphorylated; it gets inactivated after 2 h incubation; at 4 h is againphosphorylatedandintheendoftheexperimentitisagainde-phosphorylated.

A clearer picture of the effect of phenobarbital on AMPK activation is obtained when theintensity of the phosphorylation is normalized to the intensity of the total AMPK present in thesample (Figure 4-6, bottom panel). Different patterns of the time incubation effect emergewhenusing different concentrations of phenobarbital, suggesting that an analysis of the effect ofconcentrationatspecifictimepointscouldprovemoreusefultowithdrawconclusions.

Figure4-7,onpage88,showsthephosphorylationofAMPKnormalizedtothetotalamountofAMPK in the sample and in relation to the phenobarbital-free control at each time of incubation.Incubatingthecellsfor1hshowsavariationonAMPKactivationbetweena3%decreasewith10µManda3%increasewith500µMphenobarbitalincomparisonwiththecontrol.At2hincubationthevariation is the highest with an increase of 33% of AMPK phosphorylation using 250 µM ofphenobarbital, which is also observed at 6 h with an increase of 26% in comparison to thecorresponding control.With longer incubationwithphenobarbital at6h, theoverall effecton thephosphorylationofAMPKisobservedevenatlowerconcentrationsofthedrug.

ThismethodofanalysissuggeststhatphenobarbitalhasinfactaneffectonAMPKactivation,althoughveryvariableanddependentontimeofincubation.


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Figure4-6:EffectofphenobarbitalonAMPKphosphorylation(pAMPK)inMH1C1cellstreatedinserum-freemedium.MH1C1cellswereincubatedfor1h,2h,4hor6hinserum-freeDMEMcontainingphenobarbitalat0µM(lanes1–4),1µM(lanes5–8),10µM(lanes9–12),100µM(lanes13–16),250µM(lanes17–20)or500µM(lanes21–24).SampleswereanalyzedbywesternblotforAMPKandpAMPKdetection.Barplotdisplaysintensityofphosphorylated(p)AMPK(top),totalAMPK(middle)andnormalizedpAMPKintensitytototalAMPKintensityforeachsample(bottom).

Figure4-7:ComparativeanalysisoftheeffectofphenobarbitalonAMPKphosphorylationforeachtimeincubationpointinMH1C1cellstreatedinserum-freeDMEM.BarplotsdisplaythenormalizedpAMPKintensitytototalAMPKintensity(aspresentedinFigure4-6,bottompanel)inrelationtotheintensitycalculatedforthemediumcontrol(0µM)ateachtimepoint.


89

Considering the cyclic nature of AMPK’s activation over the time course of the experiment,

observed in the control samples (Figure 4-6, lanes 1 – 4, on page 88) and the variable results

obtained in thesamplesstimulatedwithphenobarbital,anewexperimentaldesign isnecessary to

eliminate thatvariability. In fact,Chingetal. 152 showed thatAMPKphosphorylation iselevated in

serum-starvedcellsincomparisontocontrolcells,whichcouldbethecausefortheunstablestateof

activation of AMPK observed in the control samples and, possibly, affecting the effect of

phenobarbitalstimulation.

AnewexperimentwasperformedinMH1C1cellsgrownandincubatedinserum-supplemented

medium.Anadditionalserum-freecontrolwasaddedinthisexperiment.

Asobservedpreviously,serum-freesamplespresentphosphorylationofAMPKinnon-treated

cells (Figure4-9, lanes1, 7, 13and19, toppanel,onpage90). Likewise, thenewcontrol samples

treated in complete medium but absent of phenobarbital stimulation also show an active AMPK

(Figure 4-9, lanes 2, 8, 14 and 20, top panel, on page 90). In fact, as shown below in Figure 4-8,

comparing the phosphorylation of AMPK normalized to the total amount of AMPK in the sample

betweencellsgrownincompleteorserum-freemediashownodistinctdifferencesthroughoutthe

incubationtimes(withtheexceptionofareductionin2hincubationincompletemedium).

Figure4-8:EffectofserumsupplementationinAMPKphosphorylation(pAMPK)fromnon-treated(0µMphenobarbital)MH1C1cells.

Bar plot displays the normalized pAMPK intensity to total AMPK intensity (as presented in Figure 4-9, bottom panel) in the different

incubationtime-points(1h,2h,4hor6h)forserum-deprivedorcompleteDMEMgrowthmedia.

TheresultsforthetreatmentwithphenobarbitalareshowninFigure4-9,onpage90.

Treatment with phenobarbital for 1 or 6 h show no marked differences on the relative

phosphorylation of AMPKwith increasing concentrations of the drug (Figure 4-9, lanes 3 – 6 and

lanes21–24,bottompanel,onpage90).IncubatingMH1C1cellsfor4hresultsinthesamerelative

phosphorylationusingthe lowestandthehighestconcentrationofphenobarbital,withareduction

onAMPKactivationobserved in themidconcentrationsutilized (Figure4-9, lanes15–18,bottom

panel, on page 90). The 2 h incubation timewith phenobarbital (Figure 4-9, lanes 9 – 12, bottom

panel,onpage90)presentssomedoseresponsebutlackslinearity;AMPKactivationislowestinthe

control,increasingwith1µMphenobarbitalandhavingamaximumvaluewith10µM,butdropping

tothesamephosphorylationvalueasthecontrolwhenusing100µMphenobarbitalandrisingagain

with500µM.

LookingattherelativeactivationofAMPKincomparisontothecontrolatthesametimepoint

(Figure4-10,onpage90)itispossibletosaythatattwohoursincubationwithphenobarbitalat10

µMproducesthehighestactivationofAMPKwhencomparedtothecontrol,buttheoverallpicture

suggeststhatthereisnodose-responseeffectofphenobarbitalonAMPKphosphorylation.


90

Figure4-9:EffectofphenobarbitalonAMPactivatedproteinkinase(AMPK)phosphorylation(pAMPK)inarathepatomacelllinetreatedincompletemedium.

MH1C1cellswereincubatedfor1h,2h,4hor6hinFBS-supplementedDMEMcontainingphenobarbitalat0µM(lanes1–4),1µM(lanes5–8),10µM(lanes9–12),100µM(lanes13–16),250µM

(lanes17–20)or500µM(lanes21–24).SampleswereanalyzedbywesternblotforAMPKandpAMPKdetection.Barplotdisplaysintensityofphosphorylated(p)AMPK(top),totalAMPK(middle)and

normalizedpAMPKintensitytototalAMPKintensityforeachsample(bottom).

Figure4-10:ComparativeanalysisoftheeffectofphenobarbitalonAMPKphosphorylationforeachtimeincubationpointinMH1C1cellstreatedinFBS-supplementedDMEM.

BarplotsdisplaythenormalizedpAMPKintensitytototalAMPKintensity(aspresentedinFigure4-9,bottompanel)inrelationtotheintensitycalculatedforthemediumcontrol(0µM)ateachtime

point.


91

Onceagain,thevariabilityoftheeffectsofphenobarbitalonthephosphorylation/activationof

AMPKanditsendogenouscyclicactivatedstateinMH1C1cellsprovesdifficulttointerprettheresults

and establish this method as a reliable phenotypic assay to measure the effect of phenobarbital

stimulation.

4.2.2.3. InductionofcytochromeP450(CYP)2B1and2B2

The ability of PB to induce expression of P450 genes has been investigated and reviewed

extensively129,153,154.ThemajorP450typesinducedbyPBinratliverareP450b(CYP2B1)andP450e

(CYP2B2)127.

Thesametreatmentofphenobarbitalinarathepatomacellline,asthepreviousexperiments,

was used to determine the effect of the drug in CYP2B1 and CYP2B2 detection by western blot.

MH1C1 cellswere treatedwith 0 – 500µMphenobarbital in completemedium for 1 hor 6 h and

unfractionatedcelllysateswereprobedwithanantibodythatdetectsbothCYP2B1andCYP2B2.The

resultsareshowninFigure4-11.

Figure4-11:EffectofphenobarbitalinCYP2B1/2detectioninarathepatomacellline.MH1C1cellsweretreatedfor1hor6hinFBS-supplementedDMEMcontainingphenobarbitalat0µM,1µM,10µM,100µMor500µM.Expectedmolecularweight:50kDa

CYP2B1 and CYP2B2 were not detected in MH1C1 cells after treatment with phenobarbital

(Figure4-11).Theblotshowstheabsenceofabandattheexpected50kDamolecularweight.Other

bands are detected with this antibody at higher molecular weights but none changes with

phenobarbital,suggestingacaseofunspecificantibodybinding.

ToevaluatethequalityoftheantibodyagainstCYP2B1andCYP2B2,differentfractionsofrat

liver isolated for anotherprojectwere loadedon the gel, and anewexperimentwithMH1C1 cells

treatedwithhigherphenobarbitalconcentrations(1mMand2mM)wasprobedsimultaneously(see

detailsinspecificmethods4.3.4TreatmentofMH1C1cellswithhighconcentrationofphenobarbital,

onpage106).ResultsarepresentedinFigure4-12,onpage92.


92

Figure4-12: Effectofhigh concentrationphenobarbital on thedetectionofCYP2B1/2 inMH1C1 cells using rat livermitochondriamatrixfractionaspositivecontrolforantibodyreactionquality.

Rat liver mitochondria were probed with an anti-CYP2B antibody and the presence of a

reactionbelowthe55kDamarkerbandconfirmsthepresenceofthisproteinandthecapacityofthe

antibodytodetect it (Figure4-12,arrow, lane4).Thisnewprotocolallowedfor thedetectionofa

faintbandinMH1C1cells(Figure4-12,lanes1–3),althoughstillabitlowerinmolecularweightas

comparedtotheratliversamples.Theresultsalsoshowthattreatmentwithphenobarbitalashigh

as2mMdoesnotincreasethedetectionofCYP2B1orCYP2B2comparedtothenegativecontrol.

Since the new method for treatment of MH1C1 cells with higher concentration of

phenobarbital revealed improvements in the detection of P450 proteins, the same protocol was

appliedforthedetectionofAMPKanditsphosphorylationstate.

Figure 4-13: Effect of high concentration phenobarbital on the detection ofCYP2B1/2andAMPKphosphorylationinarathepatomacellline.MH1C1 cells were treated with 0 mM (NaCl control), 1 mM or 2 mMphenobarbitalandanalyzedbySDS-PAGEfollowedbyWesternBlot.

The new protocol confirmed that even with high

concentrationofphenobarbital(1mMor2mM),MH1C1

cellsdonotshowdifferencesinAMPKphosphorylationin

comparisontothenegativecontrol(Figure4-13)


93

4.2.2.4. DetectionofD-aminoacidoxidase(DAO)inMH1C1cells

UnpublishedCCMSdatasuggestedD-aminoacidoxidaseasapossibletargetofphenobarbital

inratlivermitochondria.D-AminoacidoxidaseisaFAD-dependentenzymethatcatalyzesoxidative

deaminationofD-aminoacids.In1965,GiudittaandCasola155determinedthatphenobarbitalhadan

inhibitoryconstant(Ki)of4x10-3MagainstD-aminooxidase.

To determine if DAO could be used as a phenotypic assay to monitor response to

phenobarbitaltreatment,MH1C1celllysateinaserialdilutionwasprobedfortheproteinbywestern

blot.ResultsshowthatDAOisundetectableinMH1C1lysate(Figure4-14,lanes6–10),evenatthe

highestinputof50µgtotalprotein.Toconfirmantibodyspecificity,purifiedDAOwasloadedalsoin

serialdilution(Figure4-14,lanes1–5).

Figure4-14:DetectionofD-aminoacidoxidase(DAO)inpurifiedfractionsandMH1C1lysate.

Parallel experiments to determine the inhibitory action of phenobarbital on DAO were

performedatcaprotecbyDr.YanLuo.SheproducedrecombinantDAOandtestedforphenobarbital

inhibitionviaanAmplexRedbasedassay.Resultsconfirmedthatphenobarbitalaffinityislowforrat

DAO(bothrecombinantandfromlivermitochondria)withanIC50>400µMandinactiveagainstDAO

fromahumanhepatocytecellline(HepG2)withanIC50>10mM.

4.2.2.5. Effectofphenobarbitaltreatmentinratprimaryhepatocytes

Despite all efforts to develop a phenotypic assay for the effect of phenobarbital in a rat

hepatoma cell line, the resultswere so far negative or inconclusive. The genetic andphysiological

differences induced during the establishment of the cell line could be responsible for the

ineffectivenessoftheestablishedassays.Forthisreason,anewbiologicalsystem,lessmanipulated,

wasselectedtodevelopfurtherphenotypicassays:ratprimaryhepatocytes.

Theratprimaryhepatocyteswereobtained frozenandusedalways in the firstpassage.The

previously established phenotypic assays to determine AMPK phosphorylation, CAR translocation,

CYP2B1/2andDAOdetectioninresponsetophenobarbitaltreatmentwereperformedinratprimary

hepatocytes.

Theresultsoftheeffectof1mMphenobarbitalonratprimaryhepatocytesarepresentedin

Figure4-15,onpage94.


94

Figure4-15:Effectofphenobarbital inthedetectionofD-aminoacidoxidase(DAO),CYP2B1/2andAMPKphosphorylation(pAMPK)fromratprimaryhepatocytes.

As seen in Figure 4-15, rat primary hepatocytes treatedwith 1mMphenobarbital show no

differences to control regarding AMPK detection and the drug also does not promote the

phosphorylation of AMPK at its threonine 172. Similarly, CYP2B1 or CYP2B2 detection is also not

affectedbythephenobarbitalstimulus.Finally,DAOisnotdetectedineithercontrolortreatedrat

primaryhepatocytes.

The results here presented are sufficient to question the published data regardingwestern

blot methods as a readout system for the effect of phenobarbital in rat hepatocytes from both

primaryandhepatomaorigins.

Analternativemethodof detection for all thepreviouslydescribedmarkerswasdeveloped,

usingmRNAexpressionasareadoutsystem.

4.2.2.6. EffectofphenobarbitalonCYP2B1andCYP2B2mRNAexpressioninrathepatocytes

Reverse-transcription polymerase chain reaction (RT-PCR) is a well-established method to

evaluate the effect of compounds in living cells. A semi-quantitative RT-PCRmethodwas used to

compare treated samples to controls. Extracted RNA and synthesized cDNA was quantified and

loaded in the sameamountsateach step forall samples, and thehousekeepinggeneG3PDHwasusedasinternalcontroltoconfirmequalloadingbetweensamples.Detailedmethodsaredescribed

in4.3.6RT-PCR,onpage107.

Non-treatedMH1C1cellswereusedinitiallytoestablishtheRNAextractionandcDNAsynthesis

protocols, followedbyspecificamplificationof thehousekeepinggeneG3PDH.ThequalityofeachstepwasanalyzedbyagarosegelelectrophoresisandispresentedinFigure4-16,onpage95.


95

Figure4-16:AgarosegelelectrophoresisofRNA,cDNAandPCRproducts.QualitycontrolforRNAextractionfromMH1C1cells,cDNAsynthesisandPCRforthehousekeepinggeneG3PDH

AsseenonFigure4-16,lane1,totalRNAwassuccessfullyextractedfromtherathepatomacell

lineand the28Sand18S rRNAbandsareclearlyvisible.The reverse transcriptionofpoly-AmRNA

was also successful although some remaining RNA is still detected after purification of the cDNA

(Figure4-16,lane2),whichcanbespecificallydegradedbyaddingRNaseHafterthecDNAsynthesis

(Figure4-16,lane3).Thepurificationprocessdoneinasamplewithoutreversetranscriptase(Figure

4-16,lane4)showsthesameleftoverRNAbutnoadditionalimpurities.ToconfirmcDNAquality,the

specificamplificationofthehousekeepinggeneG3PDHwasdoneinbothcDNAsamples(Figure4-16,

lanes5and6)containingornotRNaseHandasingleintensebandattheexpected983bpconfirms

thatsamplesshouldbetreatedwiththeRNaseHenzymeaftercDNAsynthesis.Additionalnegative

controls(Figure4-16,lanes7and8)inpurewaterorlackingthereversetranscriptaseenzymewere

amplifiedwiththespecificprimersforG3PDHbutpresentednovisibleband,confirmingthequality

ofthesamplesandthespecificityofthePCR.

Having confirmed each step of the RNA extraction, cDNA synthesis and gene-specific PCR,

MH1C1cellstreatedwitheither1mMor2mMphenobarbitalweresubjectedtothesameprotocol.

Untreated MH1C1 cells and rat liver tissue were used as controls to monitor the expression of

CYP2B1, as was previously tested by western blot but yielded no discernible results (see Results4.2.2.3InductionofcytochromeP450(CYP)2B1and2B2,onpage91).

Figure 4-17, on page 96, clearly shows an increase of CYP2B1 mRNA expression upon

treatmentwithphenobarbital.


96

Figure4-17:EffectofphenobarbitalonCYP2B1mRNAexpressioninarathepatomacellline.MH1C1 cells were treated with 1 or 2 mMphenobarbital (PB) and one vehicle control(0.9%NaCl)over-nightat37°C(5%CO2).TotalRNAwasextractedfromthecellsandthemRNAwas specifically transcribed into cDNA using areverse transcriptase and oligo (dT)20 primers.RNA extraction and cDNA synthesis was alsodone from a rat liver sample. CYP2B1 wasamplifiedbyPCRusingspecificprimersandtheproduct was run on a 2% agarose gel in TBE.The expected amplification product is 204 bp(toppanel).ThehousekeepinggeneG3PDHwasusedasloadingcontrol(bottompanel).

RatlivertissueandMH1C1cellshavethesamelevelofCYP2B1mRNAexpression(Figure4-17,

lanes1and2,toppanel).Therathepatomacelllinewhentreatedwithphenobarbitalincreasesthe

productionofCYP2B1mRNA(Figure4-17,lanes3and4).Theresultsoftheeffectofphenobarbital

on the expression of the cytochrome P450 are confirmed by the unchanged intensity of the

housekeepinggeneG3PDHusedasloadingcontrol(Figure4-17,lane1–4,bottompanel).

This phenotypic assay was replicated and more genes were probed for changes with

phenobarbital treatment in primary rat hepatocytes. Figure 4-18, shows the effects of 1 mM

phenobarbital treatment on the expression of D-amino acid oxidase (DAO), cytochrome P450 2B1

(CYP2B1) and cytochrome P450 2B2 using two different pairs of primers that amplify different

regions of the gene (CYP2B2 and CYP2B2.1). Again, the housekeeping gene G3PDH was used asloadingcontrol.

Figure4-18:EffectofphenobarbitalonCYP2B1mRNAexpressioninratprimaryhepatocytes.Ratprimaryhepatocytes(1x106cells)wereseededina6-wellplatewithsupplementedWilliam’sEmediumandallowedtoadhereanddifferentiatefor6hours.Thecellswerethentreatedwith1mMphenobarbital(PB)and0.9%NaClvehiclecontrol(Ctrl)over-nightat37°C(5%CO2).TotalRNAwasextractedfromthecellsandthemRNAwasspecificallytranscribedintocDNAusingareversetranscriptaseandoligo(dT)20primers.TargetgeneswereamplifiedbyPCRusingspecificprimersandtheproductsrunona2%agarosegelinTBE.


97

Ratprimaryhepatocytesweretreatedwith1mMphenobarbitalandmonitoredfortheeffect

ontheexpressionofmRNAfromdifferentgenes.ThesimilarlevelofexpressionofG3PDHincontrol(Ctrl)andtreated(PB)samplesallowstoestablishthatanydifferenceobservedinotheramplification

productsisduetoamountofsample(Figure4-18,lanes1and2).

Previouswestern blot results (Figure 4-15, on page 94) had failed to elucidatewhether rat

primaryhepatocytescontainedDAO.InFigure4-18,itisclearthatthesecellsexpressDAOmRNAbut

itsexpression isnotaffectedby treatmentwithphenobarbital (Figure4-18, lanes3and4).Oneof

theanalyzed cytochromeP450mRNA (CYP2B2) didnot showa visible increase inexpressionafterphenobarbitalstimulusevenwhenanalyzingthegeneexpressionwithtwodifferentsetsofspecific

primers(Figure4-18,lanes7–10).

TheexpressionofCYP2B1mRNAfromratprimaryhepatocytes,however, isclearly increased

afterphenobarbitaltreatment(Figure4-18,lanes5and6),ashighlightedintheredbox,confirming

thesameeffectobservedpreviouslyinMH1C1cells(Figure4-17,onpage96).

These results confirm the establishment of a working phenotypic assay for the effect of

phenobarbitalinratlivercells.Thiseffectcanbemonitoredbysemi-quantitativeRT-PCRofCYP2B1mRNAexpression.ThisalsoconfirmsthatbothratprimaryhepatocytesandMH1C1cellscanbeused

toidentifyspecificbindingpartnersofphenobarbitalinlivingcellsthroughthepreviouslyestablished

CCMSusingclickchemistry.

4.2.3. CapturingphenobarbitaltargetsinMH1C1celllysate

Theinitialexperimentto identifyphenobarbitaltargetswasperformedin lysatefromtherat

hepatomaMH1C1cellline.OnlyphenobarbitalcapturecompoundsA(CC10)andC(CC12)wereused

because, as described previously, compound B (CC 11) did not yield sufficient amounts for a

comprehensivestudy.Also,eventhoughthepermeabilityofcompoundC(CC12)wasdeterminedas

beinglow(see4.2.1Determiningpermeabilityofphenobarbital-alkynecapturecompounds,onpage

84),theinitialapproachwasdesignedinlysate,wherecompoundpermeabilitydoesnotplayarole,

inordertocastawidernetfortheidentificationofputativephenobarbitaltargets.

Three separate experiments were performed (I, II and III) with varying compound and

competitor (CC 15) concentrations, as well as individual and combined data analyses. Detailed

informationontheexperimentprotocolisdescribedintheSpecificMethodssection4.3.7Captureof

phenobarbitaltargetsinMH1C1celllysate,onpage109.

Table4–2on the followingpage shows the summaryof the identificationson the individual

experiments or in combined analysis. More specifically, results were combined for both capture

compounds ineachexperimentor forall experiments toallow foran investigativeanalysison the

reproducibilityoftheCCMSoutcome.


98

Experiment I II III I+II+III

Compound CC10 CC12CC10+

CC12CC10 CC12

CC10+

CC12

CC10CC10 CC12

1µM 5µM

Totalproteinsidentified 1190 1181 1265 920 958 1013 976 2020 2020 1460

Student’st-test<0.05 123 26 18 25 175 126 55 17 2 2(%oftotal) (10%) (2.2%) (1.4%) (2.7%) (18%) (12%) (5.6%) (0.8%) (0.1%) (0.1%)

Assay/Competitionfold-change>2 139 74 133 48 41 51 33 70 146 98(%oftotal) (12%) (6.3%) (11%) (5.2%) (4.3%) (5.0%) (3.4%) (3.5%) (7.2%) (4.9%)

Competedandsignificantproteins 37 5 2 0 8 2 2 7 2 0(%oftotal) (3.1%) (0.4%) (0.2%) (0%) (0.8%) (0.2%) (0.2%) (0.3%) (0.1%) (0%)

Table4–2:SummaryofidentificationsfromphenobarbitalcaptureexperimentsinMH1C1celllysate.Threedifferentexperiments(I,IIandIII),eachconsistingofmultipletechnicalreplicates,wereanalyzedforputativetargetsoftwodifferentphenobarbitalcapturecompounds(CC10andCC12).TheseresultswereanalyzedwithMaxQuantv.1.3.0.3inanindependentmannerorincombination(symbolizedby+)

Table4–2showsthatthetotalnumberofidentificationsbetweenexperimentsvariedbetween

920 and 2020 but more strikingly that putative targets for phenobarbital, here referred to as

competedandsignificanthits,spansfromnoneto37usingthesamecompound.

An extensive analysis on each individual and combined result could be presented but the

variability of results would confound the analysis. To find the minimal common denominator all

experiments performed with the permeable compound CC10 were analyzed together, treating

samples as technical replicates, and 2 proteins are identified as significantly competed by

phenobarbital(Table4–2,I+II+III,CC10).

The first,Wdr1, also known asWD repeat*-containing protein 1, has a fold-change of 11.8

between assays and competitions with a calculated p-value of 0.024. According to Uniprot, thisprotein induces disassembly of actin filaments and is involved in platelet degranulation. Its

associationtoanycommonlyreportedphenobarbitaleffectisatfirstglancedistant,butithasbeen

publishedthatphenobarbitalincreasestheexpressionofWdr1mRNAinmouseliverphenobarbital-

induced tumorsand isdependentonCARsince the sameeffectwasnotobserved in tumors from

CAR knock-out mice (supplementary tables S3E and S3I in Phillips and Goodman 149). The direct

implicationofthisputativedirectbinderofphenobarbitalshouldbefurtherinvestigatedsinceWD-

repeatcontainingproteinsareknowntobeinvolvedinseveralimportantbiologicalfunctionssuchas

signaltransduction,transcriptionregulationandareassociatedwithdifferenthumandiseases156.It

is interesting to note that RACK1, a protein thought to have a role in phenobarbital effect 142, as

describedintheIntroductionsection,isaWD-repeatprotein.

Ybx1wasalsofoundtobesignificantlycompetedbyphenobarbitalwithafold-changeof2.6

andp-valueof 0.046. Ybx1, knownas nuclease-sensitive element-bindingprotein 1,would aswell

appear not to be related to known phenobarbital effects in a superficial analysis. However, an

alternativenameforthisprotein,CCAAT-bindingtranscriptionfactorIsubunitAsuggestsotherwise.

Asdescribedintheintroduction,CYP2Bgenespossessaphenobarbitalresponsiveenhancermodule

*WDrepeatproteinsaredefinedbythepresenceoffourormorerepeatingunitscontainingaconservedcoreofapproximately40aminoacidsthatusuallyendwithtryptophan-asparticacid(WD)


99

(PBREM)composedbyabindingsitefornuclearfactor-1(NF-1)flankedbytwoDR-4nuclearreceptor

(NR) binding sites for a heterodimer of constitutive androstane receptor (CAR) and retinoid X

receptor(RXR)132,157.Interestingly,NF-1bindstoaso-calledY-boxorCCAAT-box,muchlikeYbx1158–

160. Among several functions attributed to Ybx1, there is another association of this protein to a

knownphenobarbitaleffect,namelyregardingthesmallmolecule’sinteractionwithEGFR161.Ybx1,

alsoreferredtoasYB-1,wasshowntobindtotheEGFRpromoterandreducingYB-1levelsresulted

inthedown-regulationofEGFR162.ThecaptureofYbx1withthephenobarbitalcapturecompound

suggestsadirectbindingand,given the functionof thisprotein in the regulationof severalgenes,

thiscouldbeacontributingfactortothecellularpleiotropiceffectofphenobarbital.

Inordernottolooseinformationoneachindividualexperiment,acomparisonbetweenthe

different resultsusinga scoringmethod (aspreviouslydone in3.2.4Capturing in livingK562cells,

Table 3–8, on page 72) was used to retrieve the most relevant putative hits for phenobarbital

capture inMH1C1 lysate. Analyzing the datawith values based onMS-positive identifications (LFQ

values inMaxQuantsoftware),a totalof19hitswere identified inmore thanoneexperimentand

the fold-change and corresponding p-values are presented in Table 4–3, on page 100. This listincludes the previously describedWdr1 and Ybx1 but also reveals other interesting hits regarding

previously described associations with phenobarbital interacting mechanisms. Running a STRING

interaction analysis, two clusters of interacting proteins are revealed (Figure 4-19) and a pathway

analysisindicatesthatAMPKpathwayproteinsareenrichedinthislist,namelythedifferentsubunits

ofPP2AphosphatasethathasbeendescribedintheEGFRsignalingpathwayintheintroduction(see

Figure4-2:PhenobarbitalinducesCYP2BexpressionviaEGFRinhibition,onpage83).

Figure4-19:STRINGproteininteractionanalysisofsignificantlycompeted proteins in phenobarbital capture experiments inMH1C1lysateBlue: binding; black: reaction; pink: post-translationalmodification; purple: catalysis; green: association in curateddatabases.

Three proteins were identified in 3

different experiments as phenobarbital

binder candidates: Psmb1, Aqp1 and

Gsta3/Gsta5.Psmb1isaproteasomesubunit

that has been mentioned to increase its

mRNA expression upon phenobarbital

stimulus,butthiseffectwasonlyobservedin

asubsetoftheexperimentanditsrelevance

remains dubious 163. A surprising hit was

Aqp1, aquaporin-1, which regulates the influx of water in cells. It has been described that

phenobarbital increases and decreases Aqp1 mRNA expression and that CAR is involved in the

process164,165.Interestingly,Aqp1andthelargeraquaporinfamilyofproteinsplayarelevantrolein

epilepsy 166–168, the initial therapeutic use of phenobarbital. Lastly, glutathione S-transferase alpha

subunits (Gsta3/Gsta5)were targeted by phenobarbital in the capture experiments. This family of

proteins is involved in the protection against stress sensors in the cellular environment and its

stimulationbyphenobarbitaliswellestablishedintheliterature169.


100

Specificity(Assay/Competition) Intensityfold-change(log2) Student’st-test(pvalue)

Experiment I II* III I+II+III I II* III I+II+III compound CC10 CC12 CC10 CC12 CC10

CC10 CC12

CC10

+

CC12

CC10 CC12 CC10 CC12 CC10CC10 CC12

CC10

+

CC12

Genename 10µM 10µM 1µM 5µM 10µM 10µM 1µM 5µM Score

Aqp1 0.3 12.6 1.0 1.1 0.1 0.3 0.3 3.4 0.6 0.233 0.028 0.189 0.028 0.880 0.669 0.480 0.085 0.142 3Gsta3;Gsta5 2.8 0.1 #N/I #N/I 0.7 1.7 2.3 0.1 1.8 0.033 0.552 #N/I #N/I 0.219 0.030 0.175 0.917 0.170 3

Psmb1 12.9 0.2 #N/I -7.2 1.2 9.5 5.7 0.1 1.6 0.014 0.900 #N/I 0.423 0.627 0.036 0.100 0.933 0.189 3Aldoa 2.0 -0.8 -1.0 -0.9 0.7 1.6 1.6 -0.8 0.8 0.001 0.489 0.238 0.022 0.400 0.061 0.225 0.533 0.398 2Arcn1 0.2 -0.1 -0.3 -0.6 0.4 8.7 0.2 -0.1 0.1 0.312 0.772 0.088 0.029 0.016 0.005 0.858 0.922 0.936 2

Etfb 11.8 #N/I #N/I #N/I 9.8 9.6 10.5 #N/I 10.2 0.500 #N/I #N/I #N/I 0.003 0.188 0.082 #N/I 0.082 2Got2 3.0 0.1 -0.5 -0.6 0.9 1.3 1.9 -0.2 1.5 0.036 0.879 0.012 0.047 0.142 0.244 0.118 0.763 0.125 2

Mdh1 1.0 0.1 -0.5 -0.6 1.2 1.5 1.0 #N/I 0.6 0.247 0.961 0.041 0.035 0.048 0.092 0.415 0.997 0.464 2Me1 3.5 -1.0 -0.6 -0.9 1.4 2.5 2.4 -1.0 1.5 0.063 0.716 0.199 0.032 0.091 0.010 0.117 0.575 0.175 2Pklr #N/I #N/I -1.0 -0.7 0.8 1.3 0.6 -0.7 0.4 #N/I #N/I 0.272 0.165 0.152 0.016 0.211 0.473 0.398 2Ppp2cb;Ppp2ca 1.7 12.4 #N/I #N/I -5.6 6.5 1.6 11.0 2.5 0.255 0.046 #N/I #N/I 0.391 0.389 0.389 0.179 0.107 2Ppp2r1b -11.5 -11.1 0.6 8.7 -4.5 0.4 -2.9 -1.9 -2.4 0.500 0.500 0.702 0.001 0.391 0.391 0.340 0.509 0.224 2Rab8a;Rab8b 11.2 1.0 #N/I #N/I #N/I -12.0 11.2 1.0 2.1 0.024 0.482 #N/I #N/I #N/I 0.391 0.164 0.532 0.145 2

Rtn3 #N/I #N/I 0.6 2.4 0.8 10.2 1.0 2.4 1.2 #N/I #N/I 0.162 0.035 0.437 0.391 0.258 0.146 0.139 2Sdad1 -0.3 -0.1 1.2 7.7 #N/I 0.9 -0.3 -0.1 -0.2 0.071 0.891 0.132 0.013 0.912 0.211 0.839 0.960 0.854 2

Syngr1 -0.3 12.3 1.6 2.7 -0.9 0.4 -0.2 6.0 0.6 0.395 0.109 0.162 0.013 0.434 0.391 0.854 0.133 0.490 2Tpp2 1.5 1.4 -0.3 -0.2 1.3 1.0 1.2 1.0 1.1 0.317 0.342 0.050 0.353 0.015 0.179 0.268 0.375 0.150 2Wdr1 #N/I #N/I #N/I #N/I 12.1 11.2 11.8 #N/I 11.8 #N/I #N/I #N/I #N/I 0.001 0.391 0.024 #N/I 0.026 2

Ybx1 #N/I #N/I -6.7 5.3 7.9 3.3 2.6 4.6 2.7 #N/I #N/I 0.423 0.423 0.067 0.023 0.041 0.374 0.038 2Table4–3:Potentialhitsforphenobarbitalinarathepatomacellline(MH1C1)from3individualexperimentsusingaMS-basedanalysis

(I-III)eachconsistingof2–4replicatesforassaysandcompetitionsusingtwopre-clickedphenobarbitalcapturecompounds(CC10andCC12).Valuesinboldmarkspecific(log2fold-change>1)andsignificant(p

value<0.05)resultsforagivenexperiment.Scoreiscalculatedbythesumofnumberofresultsthatfulfilledboththecriteriaofspecificityandsignificance.#N/I:notidentified.*CC10subsetofexperimentIIwas

analyzedindividuallywithMaxQuantsoftwareandwasaddedtothescorecalculations.


101

4.2.4. CaptureofphenobarbitaltargetsinlivingMH1C1cells

The capture of phenobarbital targets in living cells was done to improve upon the alreadysuccessful results obtained in the capture experiments from lysate samples and establish a newmethodfortheidentificationofsmall-moleculebindingpartnersthatbetterresemblesthedeliverysystemofdrugsinlivingorganisms.

TheexperimentwasdonewithlivingMH1C1cellsusingtriplicatesofassaysandcompetitionsandthedetailedprotocolisdescribedintheSpecificMethodssection4.3.8CaptureofphenobarbitaltargetsinlivingMH1C1cells,onpage110.

Table4–4gathersthesummaryoftheidentificationsineachsubsetoftheexperiment.

Pre-clicked Click

Alignment-based

MS-based Alignment-based

MS-based



41(5.5%)

43(5.9%)

52(3.8%)

44(3.2%)


16(2.2%)

33(4.5%)

21(1.5%)

49(3.6%)


2(0.3%)

1(0.1%)

1(0.1%)

1(0.1%)

Table4–4:SummaryofidentificationsfromphenobarbitalcaptureexperimentsinMH1C1livingcells.

ListofproteinswasobtainedusingMaxQuantv1.3.0.3.Numbers for identificationsbasedonspectralcounts (MS-based)andalignment-

basedaregiven.

One immediate observation of Table 4–4 is that a larger number of proteins is identified in

samples where the click reaction was done after the photo-crosslink to the target, 740 vs. 1363where the capture compoundwaspre-clicked to thebiotin anchorduring the cell incubation. Thedifference innumberscouldbeexplainedby theprobable impermeabilityof the largerpre-clickedcompound,preventing it todiffuse into thecelland thereforemainly targetingsurfaceproteinsoronlyafterendocytosis.Adifferentperspectivesuggeststhatthe largernumberof identifications inclick samples could be due to unspecific capture inherent to the CuAAC reaction, although initialexperiments(describedinChapter2.Labelingaproteinusingclickchemistry)showthattheCuAACisrathercleanbutwithlowyields.

Even thougha largenumberofproteinswere identified,only fourpass thecompetitionandreproducibilitycriteria.ThevaluesfortheseputativephenobarbitalbindersarepresentedinTable4–5,onthefollowingpage.


102

Specificity(Assay/Competition)

Protein TotalMScounts Alignment-based MS-based

Genename

Assay Comp. Intensityfold-change(log2)


Intensityfold-change(log2)


Pre-click

Them6 11 5 1.6 0.002 1.4 0.014

Anxa4 1 1 1.2 0.022 9.6 0.423

Click

Arf5 3 0 4.5 0.032 -7.9 0.423

Dnm2 3 0 0.1 0.761 12.7 0.008Table4–5:ListofcompetedandsignificanthitsinphenobarbitalcaptureexperimentsinMH1C1livingcells.

TotalMScountsfromtechnicalreplicates(n=3)ofassayandcompetition(comp.)samples.Intensityfold-changeandsignificancelevelsare

givenforspecificity(assayversuscompetition)ofhits.ValuesinboldindicateeitherFC>2orpval<0.05.

The list ofproteins identified in the captureexperimentusingphenobarbital in livingMH1C1

cells, presented in Table 4–5, do not coincide with the previously described putative bindersobtainedwiththecaptureinlysateofthesamecellline.Thereis,however,acommondenominatortoalltheseidentifiedproteins:EGFR.

Them6isaratheruncharacterizedthioesterasewithonlysevenreferencesonPubmedand9known protein interactions. The thioesterases, or thioester hydrolases, comprise a large enzymegroupwhosemembershydrolyzethethioesterbondbetweenacarbonylgroupandasulfuratom.Interestingly, Them6 has been co-immunoprecipitated with EGFR 170 and was also previouslyidentified as a potential phenobarbital target in Experiment B of the capture in MH1C1 lysatedescribed in the previous paragraph (the alignment-based values for Them6 revealed a log2 fold-changebetweenassaysandcompetitionsof1.3withanassociatedp-valueof0.002).

AnnexinA4,Anxa4,isacalcium/phospholipid-bindingproteinthatpromotesmembranefusionand is involved in exocytosis. Anxa4 has clinical significance in cancer 171, and even if this specificmemberof theannexin familyhasnotbeendescribed inEGFRtransductioncontext, severalotherannexinshavebeenassociatedwithendocytosisandEGFRsignaling172,173.

Arf5,alsoknowasADP-ribosylationfactor5isaGTP-bindingproteinthatisInvolvedinproteintrafficking174andrecentlyfoundtoco-localizewithEGFRinA431cells175.

Dnm2,Dynamin-2, isamicrotubule-associatedforce-producingprotein involved inproducingmicrotubulebundlesandabletobindandhydrolyzeGTP.Dnm2playsanimportantroleinvesiculartrafficking processes, in particular endocytosis 176. Association of Dnm2 to EGFR has also beendescribedmultiplepublicationsasanEGF-inducedEGFR-associatedprotein177,178.

Onceagain,resultsseemtopointtowardsaconnectionwithEGFR,inlinewithMutho’srecentfindings142,butthequestionthatremainsiswhyisn’tEGFRcapturedintheseexperiments.Arethesethe true phenobarbital binders that establish the connection between EGFR signaling andphenobarbitalphenotypiceffectorarethese interactingproteinscapturedduetounspecificcross-linking to neighboring proteins inherent to the design of capture compounds? It can also bepostulated that EGFR peptides are not identified in the CCMS experiments because the attachedcapturecompoundaltersthemassofthepeptidesandaffectstheanalysisinthedatabasesearch.


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4.2.5. CapturingphenobarbitaltargetsintissuewithhighEGFRexpressionWithpilingevidencethatphenobarbitaltargetsproteinsrelatedtoEGFR,andgiventherecent

publicationofMutohandco-workers142thatconfirmsEGFRasatargetforphenobarbital, itwouldbeinterestingtoverifyifcaptureexperimentsdeterminethereceptorasaprimarybinder,oriftheassociatedproteinsidentifiedinthepreviousparagraphsarestillpreferentialtothedruginatissueexpressinghighlevelsofEGFR.

According to the Human Protein Atlas, a tissue-based map of the human proteome 179,trophoblasticcellsfromplacentapresentahighexpressionofEGFRprotein.

Humanplacentalysatewasusedassampletoidentifyphenobarbitaltargetsusingacompletepre-clicked phenobarbital capture compound. Detailed protocol is described in Specific Methodssection4.3.9Captureofphenobarbitaltargetsinhumanplacentalysate,onpage111.

Table 4–6: Summary of identifications from

phenobarbital capture experiment in human

placentalysate.

List of proteins was obtained using MaxQuant

v1.3.0.3. Numbers for identifications based on

spectralcounts(MS-based)andalignment-based

aregiven.

From over 2000 protein identifications, only 6 were competed by phenobarbital in areproduciblemanner. These identifications, summarized in Table 4–6, yield different resultswhenanalyzing the values basedon alignment features or exclusively on sequencedpeptides,with onlyonesignificantlycompetedtargetidentifiedinbothmethods.

The list of significantly competed proteins with phenobarbital in human placenta lysate ispresentedinTable4–7.

Specificity(Assay/Competition)

Protein TotalMScounts Alignment-based MS-based

Genename

Assay Comp. Intensityfold-change(log2)


Intensityfold-change(log2)


SRP72 52 37 2.4 <0.001 0.2 0.087

YBX1/3/5 4 3 1.5 0.001 13.3 <0.001

MICALL1 4 1 1.7 0.011 1.4 0.378

TMEM97 3 4 1.1 0.045 1.3 0.073

MESDC2 3 1 0.1 0.673 12.4 <0.001

GAK 3 2 0.8 0.047 12.2 <0.001

EGFR 33 30 0.1 0.761 0.14 0.129Table4–7:Listofcompetedandsignificanthitsinphenobarbitalcaptureexperimentsinhumanplacentalysate.

TotalMScountsfromtechnicalreplicates(n=3)ofassayandcompetition(comp.)samples.Intensityfold-changeandsignificancelevelsare

givenforspecificity(assayversuscompetition)ofhits.EGFRisaddedseparatelyforfailingcriteria.Values inbold indicateeitherFC>2or

pval<0.05.

Pre-click

Alignment-based

MS-based

Totalproteinsidentified 2054 2047


80(3.9%)

64(3.1%)


51(2.5%)

78(3.8%)


4(0.2%)

3(0.1%)


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The first observation of the phenobarbital capture experiment in a tissue expressing highlevelsofEGFR is that thereceptor is indeedcapturedbut thecompetitionwith freephenobarbitalcompetitor does not exceed the two-fold ratio between assays and competitions, nor is theconfidenceleveloftheidentificationsacceptable.

Interestingly,theonlypositiveidentificationusingeitheranalignmentorMS-basedanalysisisYBX,thepreviouslydescribedY-box-bindingproteinidentifiedasatargetinrathepatomacelllysate.The results in placenta do not allow for a definite identification of the YBX isoform since thequantifiedpeptide is common toYBX1,YBX3andYBX5.Data suggests it tobeYBX1, justas in theMH1C1celllysateexperiment,becauseanadditionalYBX1peptidewasidentifiedalthoughwithoutasignificantcompetitioneffect.

The other identified putative targets for phenobarbital are also described to have anassociation toEGFR,with theexceptionof the signal recognitionparticle subunit SRP72.MICALL1,MoleculeInteractingwithCasL(MICAL)-like1protein,hasbeendescribedtobeinvolvedinreceptor-mediated endocytosis and, in complex with Rab protein, regulates EGFR trafficking 180. Cyclin G-associatedkinase(GAK)isaserine/threoninekinasewitharelevantroleincellulartrafficofclathrin-coated vesicles and it was shown that its down-regulation resulted in an enhancement of EGFRsignaling181.AlthoughnotdirectlyassociatedwithEGFR,MESDC2isachaperoneproteinspecificallyassisting thebeta-propeller/EGFmoduleswithin the familyof low-density lipoprotein receptors 182andtransmembraneproteinTMEM97wascorrelatedwithEGFRinastudyfromTongetal.178.

Toovercomethe incongruousresultsobtainedwhenusingadifferentvalueoutputfromtheMaxQuant analysis software and the way each contributing peptide generates the intensity for awholeprotein, theexperimentaldataobtained fromthephenobarbital capture inhumanplacentalysate was analyzed at the peptide level. Out of 12188 identified peptides only 710 (6%) werecompetedandoftheseonly26(0.2%)wereidentifiedwithapvalueoflessthan0.05.

These 26 unique peptides,peptides corresponding exclusivelyto a specific protein, weresubmitted to the proteininteraction database STRING andrevealed an intricate network thatshows EGFR and YBX1 as centralnodes(Figure4-20).

Gathered evidence suggeststhat phenobarbital binds primarilytoYBX1and there is ahighaffinitytowards proteins associated toEGFRsignaling.

Figure 4-20: STRING protein interaction analysis

of significantly competed proteins in

phenobarbital capture experiments in human

placentalysate.

Blue: binding; pink: post-translational

modification;purple:catalysis;green:association

incurateddatabases.

Chapter4:Phenobarbital–newbindersforanolddrug|SpecificMethods

105

4.3. SPECIFICMETHODSFORTHEPHENOBARBITALSYSTEM4.3.1. Structuresofusedcompounds

CC10:ClickablephenobarbitalalkynecapturecompoundA(containsselectivityandatrifluoromethyl-phenyl-diazirinephotoreactivefunctions)

CC11:ClickablephenobarbitalalkynecapturecompoundB(containsselectivityandamethyl-diazirinephotoreactivefunctions)

CC12:ClickablephenobarbitalalkynecapturecompoundC(containsselectivityandaazido-tetrafluorobenzenephotoreactivefunctions)

CC15:Phenobarbitalcompetitor



106

4.3.2. TreatmentofMH1C1cellswithphenobarbital

TherathepatomacelllineMH1C1wastreatedwithincreasingconcentrationofphenobarbitalfor theanalysisofCAR translocation,AMPKphosphorylationandCYP2B1/2B2analysisbyWesternBlotting.

MH1C1 cells in DMEM (10% FBS, 1% pen/strep) were seeded in 6-well plates at 2 x 106cells/well and incubated over-night at 37 °C (5% CO2). Confluent cells were starved over-night byremovingthemediumandaddingserum-freeDMEM.

Phenobarbitaldiluted inDMEMwasaddedtothewellsatdifferentconcentrations:0,1µM,10µM,100µM,250µMand500µM.Plateswereincubatedat37°C(5%CO2)for1h,2h,4hor6h.

Aftertreatment,themediumwasremovedandthecellswerewashedwith1mLD-PBS.Cellswerescrapedin1mLoffreshD-PBSandtransferredto1.5mLmicrotubesforcellularfractionation(asdescribedin4.3.3SubcellularfractionationofMH1C1cells,below).

The cellular fractions and the unfractionated samples were resolved on a SDS-PAGE andtransferred toanitrocellulosemembrane for specificantibodyprobingasdescribed in thegeneralmethodssection(6.2BiochemistryGeneralMethods,onpage118).

4.3.3. SubcellularfractionationofMH1C1cells

MH1C1cellpelletsfromculturein6-wellplatesforphenobarbitaltreatmentwerefractionatedusingtheNE-PER®NuclearandCytoplasmicExtractionkit(PierceBiotechnology™).

Cells were scraped in 1 mL D-PBS and transferred to 1.5 mL microtubes to pellet bycentrifugationat500xgfor3min.Thesupernatantwascarefullyremovedwithamicropipetteanddiscarded,leavingadrypelletwith≅15µLofpackedvolume.150µLofice-coldCERIreagentwasadded to thecellpelletand the tubewasstirred inavortexatmaximumspeed for15seconds toresuspendthecells.Thetubewasincubatedonicefor10min.8.25µLofice-coldCERIIreagentwasadded to the tube, following 5 seconds vortex and 1 min incubation on ice. The tube was againvortexed for 5 seconds and centrifuged for 5 min at 17.000 xg. The supernatant containing thecytoplasmicfractionwastransferredtoacleanpre-chilledtubeandstoredat-80°C.Theremainingpellet, containing thenuclei,was resuspended in75µLofNER reagent. The tubewas vortexedatmaximumspeedfor15secondsevery10minforatotalof40min,keepingitoniceinbetween.Thetubewascentrifugedfor10minat17.000xgandthesupernatantcontainingthenuclearextractwastransferredtoapre-chilledcleantubeandstoredat-80°C.

4.3.4. TreatmentofMH1C1cellswithhighconcentrationofphenobarbital

MH1C1 cells in DMEM (10% FBS, 1% pen/strep) were seeded in 6-well plates at 2 x 106cells/well and incubated over-night at 37 °C (5% CO2). Confluent cells were starved for 2.5 h byremovingthemediumandaddingserum-freeDMEM.Phenobarbitaldiluted in0.9%NaClata finalconcentrationof0,1mMor2mMwasadded to thewellsand incubatedover-nightat37 °C (5%CO2).Themediumwasremovedandthecellswerewashedwith1mLD-PBS.

To the wells for protein analysis, 100 µL of 2X Laemmli Buffer (containing benzonase) wasaddedtodisrupt thecells.Thesampleswere transferred tomicrotubes, sonicatedandboiled,andstored at -20 °C until analysis by SDS-PAGE and Western Blotting (as described in the generalmethodssection(6.2BiochemistryGeneralMethods,onpage118).

ForRT-PCRanalysis,thecellswereprocessedasdescribedin4.3.6RT-PCR,onpage107.


107

4.3.5. TreatmentofratprimaryhepatocyteswithphenobarbitalRatprimaryhepatocyteswereplatedasdescribed intheGeneralMethodssection(6.1.4Rat

primaryhepatocytesculture,onpage117).The cells were incubated over-night with 1 mM phenobarbital diluted in 0.9% NaCl. The

mediumwasremovedandthehepatocyteswerewashedwith1mLD-PBS.To the wells for protein analysis, 100 µL of 2X Laemmli Buffer (containing benzonase) was

addedtodisrupt thecells.Thesampleswere transferred tomicrotubes, sonicatedandboiled,andstored at -20 °C until analysis by SDS-PAGE and Western Blotting (as described in the generalmethodssection(6.2BiochemistryGeneralMethods,onpage118).

ForRT-PCRanalysis,thecellswereprocessedasdescribedbelow.

4.3.6. RT-PCRReverse-transcription polymerase chain reaction (RT-PCR) is a commonly used technique to

semi-quantitatively measure the expression of specific genes. RNA is converted into acomplementary DNA (cDNA) library that can then be probed for target genes using appropriateprimers.Oneoftheadvantagesofthistechniqueistheneedforverylittlesamplequantitytodetectthetargetsofinterest.

4.3.6.1. RNAextractionusingRNeasyProtectMiniKit

RNAwasextracted fromeitherMH1C1cellsor ratprimaryhepatocytes inculture, treatedornotwithphenobarbital,usingacommerciallyavailablekitfromQiagen™(RNeasyProtectMiniKit).

1 x 107 cells were collected to a 15 mL Falcon tube and lysed in 600 µL RLT buffer(supplementedwithDTT).Thetubewasvortexedandthelysatewashomogenizedbypassingitfivetimesthrougha20-gaugeneedleattachedtoaRNA-freesterilesyringe.600µLof70%ethanolwasaddedtothetubeandmixedbypipetting.ThesolutionwasdividedandtransferredtotwoRNeasyspin columnsplaced in2mLmicrotubes. The sampleswere spundown for30 s at10.000xg. Theflow-throughwasdiscardedand700µLofbufferRW1wasaddedtothespincolumn.Thetubeswerecentrifuged 30 s at 10.000 xg. 500 µL of RPE buffer was added to the spin column and againcentrifuged30sat10.000xg.Fresh500µLofRPEbufferwasaddedto thecolumnandthe tubesspundownfor2minat10.000xg.Thespincolumnwastransferredtoaclean2mLmicrotubeandspundownfor1minatmaximumspeed(16.000xg).Thespincolumnwasplacedinaclean1.5mLmicrotubeandRNAwaselutedwith40µLofRNAse-freewater.Thecolumnwascentrifuged1minat10.000xg toelutetheRNA.Samplesweresnapfrozen in liquidnitrogenandstoredat -80°Cuntilfurtheruse.


108

4.3.6.2. RNAquantificationandpuritycalculation

Nucleic acids have absorbancemaxima at 260 nm. Historically, the ratio of this absorbancemaximumtotheabsorbanceat280nmhasbeenusedasameasureofpurityinbothDNAandRNAextractions.A260/280ratioofapproximately1.8isgenerallyacceptedas“pure”forDNA;aratioofapproximately2.0isgenerallyacceptedas“pure”forRNA.

ExtractedRNAfromMH1C1cellsandratprimaryhepatocyteswasdilutedinPBS(pH7.4)andthe absorption at 260 nm and 280 nm was measured in a Varian Cary 50 Bio UV-VisSpectrophotometer.

ThepurityofRNAwascalculatedaccordingtotheformula:"#$%('()*+,)."#$%(/+(01)"#2%('()*+,).

TheconcentrationofRNAwasdeterminedby:

3 = "#$%('()*+,)."#$%(/+(01)56 ×89:;<9=>?@A<=B.

TheobtainedRNAconcentrationsfromdifferentextractionsvariedbetween0.5µg/µLand1µg/µLwithapurityalwayshigherthan2.

4.3.6.3. cDNAsynthesisfromextractedmRNA

1µgoftotalRNAextractedfromMH1C1cells,ratprimaryhepatocytesorrat livertissuewasreverse-transcribedintocDNAusingtheSuperscriptIIIreversetranscriptasekitfromInvitrogen™.

1µLofoligo(dT)20primersat50µMwasmixedwith1µgoftotalRNA,1µLofdNTPmixat10mM and water to normalize volume to 13 µL. The mixture was heated for 5 min at 65 °C in athermomixerandplacedoniceforover1min.Thetubeswerespundownandadded4µLof5xFirstStrandBuffer,1µLofDTTat0.1Mand1µLSuperScriptIIIreverse-transcriptase(at200U/µL).Thesamplesweremixedby gentlypipettingand incubated for60minat 50 °C in a thermomixer. Thereactionwasinactivatedbyheating15minat70°C.1µLofRNAseHwasaddedfor20minat37°CinsomecasestoremoveRNAcomplementarytothefirststrandcDNA.

4.3.6.4. PolymeraseChainReaction(PCR)ofspecificgenes

ThecDNAlibraryfromMH1C1cells,ratprimaryhepatocytesorrat livertissue,treatedornotwithphenobarbital,wasusedastemplatetosearchforspecificgenes:CYP2B1 (usingtwodifferentpairsofprimers),CYP2B2,DAOandG3PDHasahousekeepinggenecontrol.Thesequencesfortheprimersaredescribedbelow:

Forwardprimer Reverseprimer Expectedproduct

G3PDH 5’-TGAAGGTCGGTGTCAACGGATTTGGC-3’ 5’-CATGTAGGCCATGAGGTCCACCAC-3’ 983bp

CYP2B1 5’-CTGTGGGTCATGGAGAGCTG-3’ 5’-TCACACCGGCTACCAACCCT-3’ 201bp

CYP2B2 5’-CTGTGGGTCATGGAGAGCTG-3’ 5’-TCTCACAGGCACCATCCCT-3’ 201bp

CYP2B2.1 5’-GCTCTCCTTGTGGGCTTCTT-3’ 5’-AGGACTCACTTTCTCCATGCG-3’ 804bp

DAO 5’-ATCCTGCTGGAGACAGGTTC-3’ 5’-TCTCCTCCCATCACTCCATC-3’ 1510bp


109

The optimization of the different parameters of PCR, such as cDNA input amount, MgCl2concentration, primer concentration, and number of cycles, was done according to the Taguchimethod183.

Thefinalparametersusedfortheexperimentspresentedintheresultssection(4.2.2.6EffectofphenobarbitalonCYP2B1andCYP2B2mRNAexpressioninrathepatocytes,onpage94)were:

cDNA F.primer R.primer

10xPCRbuffer

dNTPmix

T.aqpolymerase MgCl2 H2O

Volume(µL) 1.5 2 2 5 1 0.2 1.5 38.3

4.3.6.5. Agarosegelelectrophoresis

RNAqualityandPCRproductswereanalyzedbyhorizontalagarosegelelectrophoresis.0.8%–1.2%(w/v)agarosegelswerepreparedbymixingtheweighedamountofagarosein50

mL of Tris/Borate/EDTA (TBE) buffer. The solution was boiled in a microwave and cooled downbeforeadding3µLofSYBR®Safedyeandcastingontotheelectrophoresistray.

ThetraycontainingthegelwasimmersedintheelectrophoresisunitfilledwithTBEbufferandsamplesmixedwith6xloadingdyewereloadedinthewells.AppropriateDNAladderswereaddedasreference.Thegelelectrophoresiswascarriedoutfor45minat100VandbandsvisualizedusingaUVtrans-illuminatorinanImagingSystem(SyngeneG:BOXXT4).

4.3.7. CaptureofphenobarbitaltargetsinMH1C1celllysate

MH1C1 cell lysate was prepared as described in the GeneralMethods (6.1.3.1MH1C1 lysatepreparation,onpage117).

Fullpre-clickedcapturecompoundswerepreparedapriori:500µMofbiotincopper-chelating-azide(CC7)wasequilibratedwith5mMofCuSO4for10minatR.T.andthenreactedwith500µMofphenobarbital-alkyne capture compound (CC 10 or CC 12) in PBS at pH 8.5 by adding 2.5mM ofsodiumascorbate.Thereactionwasshakenvigorouslyfor10minatR.T.

400µgofMH1C1celllysatewasdistributedin200µLPCR-tubestripsandeither330µMor1mM of phenobarbital competitor (CC 15) was added to the competition samples or thecorresponding volumeofDMSOwas added to the assay samples. The vialswere incubated for 30minoniceandthenadded5µMor10µMofpre-clickedphenobarbital-biotincapturecompound.Allsampleswere incubated for60min rotatingat4 °Cand then irradiated for15minat310nm inacaproBox™ to promote photo-crosslinking of the compounds to the binding targets. Streptavidin-coatedmagneticbeadswerewashedwithPBSandincubatedwiththesamplesfor1hrotatingat4°C. The beads were recovered using a caproMag™ and then washed six times with wash bufferfollowedbythreetimeswashwith80%acetonitrileandafinalwashwithMS-gradewater.



110

4.3.8. CaptureofphenobarbitaltargetsinlivingMH1C1cells

MH1C1 cells were seeded on 6-well plates in DMEM, supplemented with 10% FBS and 1%penicillin/streptomycin, and grown to 85% confluence in a cell incubator at 37 °C (5% CO2). ThemediumwasremovedandthecellswerewashedwithD-PBStostarvewithserum-freeDMEMfor2hat37°C(5%CO2).

A pre-clicked phenobarbital capture compound (CC 10)with a biotin-copper chelating azide(CC7)wasproducedapriori:500µMofbiotincopper-chelating-azide(CC7)wasequilibratedwith5mM of CuSO4 for 10min at R.T. and then reacted with 500 µM of phenobarbital-alkyne capturecompound (CC 10) in PBS at pH 8.5 by adding 2.5mMof sodium ascorbate. The vialwas shakenvigorouslyfor10minatR.T.

2mM of phenobarbital competitor (CC 15) or equivalent volume of DMSO diluted in freshmediumwasaddedtothecompetitionwellsor totheassay, respectively.20µMofphenobarbitalcapturecompound(CC10)asisorpre-clickedtobiotin(CC10-CC7)wasthenaddedtothewells.Theplates were returned for 35 min to the cell incubator. The medium was removed and the cellswashedwithfreshD-PBS.TheplateswereplacedinaCaproBox™andirradiatedfor4minat310nm.Thecellswererecoveredtotubeswithscrapingandpelletedfor10minat400xg.

Thecellstreatedwiththepre-clickedcompoundwerelysedfor30minshakingvigorouslywith1 mL of RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% IGEPAL CA-630, 0.5% Sodiumdeoxycholate, 0.1% SDS) supplemented with 0.2 mM DTT, 0.5 µL/mL benzonase and proteinaseinhibitors.Thesamplesincubatedwiththeunclickedphenobarbitalcapturecompoundwerelysedin500µLof Lysis Buffer E as described in 6.1.3.1MH1C1 lysatepreparation, onpage117. The crudelysates from both preparations were transferred to 500 µL eppies and centrifuged for 30 min at17.000r.p.m.toremovecelldebris.

The CuAAC reaction was performed in the samples containing the unclicked capturecompoundbyadding5µMofbiotincopper-chelating-azide(CC7)(pre-equilibratedwithtwo-timesexcessCuSO4), 10µMBTTE (CC18) pre-equilibratedwith two-timesexcessCuSO4, and2.5mMofsodiumascorbate.Theclickreactionranfor30minshakingatroomtemperature.

Streptavidin-coatedmagnetic beadswerewashedwith PBS and incubatedwith the samplesfor30minrotatingat4°C.ThebeadswererecoveredusingacaproMag™andthenwashedsixtimeswithwashbufferfollowedbythreetimeswashwith80%acetonitrileandafinalwashwithMS-gradewater.



111

4.3.9. CaptureofphenobarbitaltargetsinhumanplacentalysateHuman placenta tissue was purchased from in.Vent, Berlin-Hennigsdorf, and was obtained

accordingtorelevantethicalguidelineswithinformedconsentofdonor. 400 µL of human placenta lysate, containing around 2000 µg protein, from the BioBank at

caprotec bioanalytics, were incubated with phenobarbital competitor (CC 15) at 2 mM finalconcentrationorwithequivalentvolumeofDMSOincaseofcontrolsamplesand5XCaptureBuffer.Thesampleswereequilibratedfor20minrotatingat4°C.

Fullpre-clickedcapturecompoundswerepreparedapriori:500µMofbiotincopper-chelating-azide(CC7)wasequilibratedwith5mMofCuSO4for10minatR.T.andthenreactedwith500µMofphenobarbital-alkyne capture compound (CC 10) in PBS at pH 8.5 by adding 2.5 mM of sodiumascorbate.Thereactionwasshakenvigorouslyfor10minatR.T.

Streptavidin-coated magnetic beads were pre-loaded with the pre-clicked phenobarbitalcapturecompoundusingexcesscapturecompoundtosaturatethebeads,shakingfor5minatroomtemperature.TheexcessunboundcapturecompoundwasremovedandthebeadswerewashedtwotimeswithCaptureBuffer.

The pre-loaded magnetic beads with phenobarbital capture compound were added to thesamplesand incubatedfor2hrotatingat4°C.ThebeadswerethencollectedusingacaproMag™andresuspendedin100µLofCaptureBuffer.Thesuspendedbeadswereirradiatedfor4min(twotimes2min,shakingthetubesinbetween)at310nminacaproBox™topromotephoto-crosslinking.ThemagneticbeadswererecoveredusingacaproMag™andthenwashedsixtimeswithwashbufferfollowedbythreetimeswashwith80%acetonitrileandafinalwashwithMS-gradewater.


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5. ConclusionsandOutlookThe current challenges in drug discovery have strengthened the development of new

technologies for targetdeconvolution inanunbiasedapproach to identifyunwantedoff-targetsorreveal a new path for development early in the pipeline. Recent developments in chemicalproteomics associated to advances in quantitativemass spectrometry have enabled the detectionandquantificationoflowabundantbindersordrug-inducedchangesinproteinexpressionwithlargeproteomecoverage.

Capturecompoundmass spectrometry (CCMS) introducedanoveldesign for smallmoleculeprobesthatallowtheidentificationoflowabundantandloweraffinitytargetsinacomplexbiologicalsample. The capture compounds traditionally consist of a selectivity function carrying the smallmoleculeunderstudythatisattachedtoascaffoldbearingatagforvisualizationorenrichment,suchas a fluorophoreorbiotin, andalso aprotrudingphoto-reactivemoietywithenoughdistanceandflexibilityfromthecentralcoretocovalentlybindtheprobetothesurfaceofthetargetprotein.

One of themajor limitations of capture compounds in their originally published design andotheraffinity-basedprobes is thatmost cannotpermeate through cellsdue to their size, reducingthe application to cell-surface targets or the need to prepare lysates if the aimed protein isintracellular.Thedrawbackofworkingwithproteinlysatesisthatnotallcellularproteinsareeasilysolubilized,thusexcludingpartoftheproteome,andthelysisprocessmightinfactalterthenativeconformation of proteins, which can affect the interaction with the drug molecule. Subcellularfractionation instead of full lysate has been developed as a strategy to overcome some of theseobstacles.

Bio-orthogonalchemistrywasinitiallydevelopedtostudypost-translationalmodificationsbutquicklyexpandedtoabroaderusetostudydynamicsandfunctionofbiomolecules.Severalreactantpairshavebeendevelopedbuttheazideandalkynereactionstillremainthebettersuitedforlivingcellapplicationsduetotheirreducedsize.Thebiggestdisadvantageofthisbio-orthogonalligationistheneedtoaccelerateitwithmetals,copperbeingthemostwidelyused,whichcaninducestressincells.Advancesinthetechnologyweremadewiththeintroductionofchelatingagentsthatimprovethereactionkineticsandreducethemetal-inducedstress.

Establishinganewdrugdeconvolutionmethodthatallowstheidentificationoftargetsintheirnatural cell environment was set as a primary goal in this research project. The strategy was tocombinethestrengthofcapturecompounds intheenrichmentof lowabundantand loweraffinitytargetswiththeversatilityofbio-orthogonalligationchemistry.Thenewprobedesignwouldallowitto be small enough and increase cell permeability to target intracellular proteins and specificallycapture them with a controlled chemical click reaction. One main advantage of such a workflowwould be to identify drug-binding proteins directly under conditions that are used in phenotypicscreening-baseddrugdiscovery.

A modified capture compound bearing cAMP as selectivity function and a photo-reactivityfunction,buthavingareactivealkyneinsteadofthetraditionallabelingfunction,wassynthesizedtoestablishcopper-assistedazido-alkynecycloaddition(CuAAC)clickchemistryinthecontextofCCMS.The optimization steps to establish the click reaction conditions were initially established in asolutionofrecombinantPKARI,thesubunitofproteinkinaseAthatbindscAMP.Abiotin-azidewas


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used as label to monitor this reaction and results were analyzed by western blot. The initialexperiments confirmed the specific nature of the azido-alkyne cycloaddition where no signal wasobservedinsampleslackingthecapturecompound(Figure2-2,onpage32)butalsoshowedthatthesignal was a fraction of the one obtained with a classical cAMP capture compound. DifferentparametersfromtheCuAACreactionweremodifiedtoimprovethelabelingofPKARIsuchascopperand ligands concentrations; time and pH of reaction; biotin-azide and alkyne capture compoundconcentrations; and a new fluorescent label that promised to be better suited for the labeling ofcomplexsamples,suchascellularlysates.TheoptimizationeffortsyieldedsomeimprovementfromtheinitialexperimentsbutstilldidnotachievethesameresultsaswiththeclassicalcAMPcapturecompound.

Capturing PKA subunits from cell lysates with the optimized parameters proved to be sub-optimalwhenusing thenewclickworkflowandcomparing it to resultsobtainedwith theclassicalcAMP capture compound. Although the results suggested an enrichment of PKA-related peptides,theintensityratiobetweenassaysandcompetitionsandtheirstatisticalsignificancedidnotallowfora conclusive analysis on the performance of the method. The lack of competition in severalexperiments proved to be a challenge if further efforts would be made to resolve this issue,especiallywhenthesubsequentstepwouldbetoestablishtheclickcaptureinlivingcells,wherethenatural concentration of cAMP is very high. Nevertheless, the system was useful to establishtechnical aspects of the click reaction in the context of the use of capture compounds. However,becauseoftheimpermeabilityofcAMPassuch,adifferentselectivityfunctionwasrequiredforthenextsteps.Withthisinmind,anewnon-naturalselectivityfunctionwasusedfortargetidentificationincelllysatesandlivingcellsusingthenewclickreaction.

ThekinaseinhibitordrugDasatinibwasusedforitsclinicalrelevanceandwell-knowntargets.Concomitantly to this newalkynedasatinib capture compound, a newazidewas alsoproduced incollaborationwiththegroupofTaran,fromtheCEASaclay,memberoftheBioChemLigconsortium.Thenewazide,bearingthebiotinorfluorescentTAMRAastags,alsohadacopper-chelatingmoietythatbetteracceleratedthecycloadditionreaction.Thewellcharacterizedtargetprofileofdasatinibwaswellsuitedasreferencetojudgetheeffectivenessofthenewapproach.Apreviouslypublishedstudyonlivingcellcapturewithadasatinibprobefailedtoproduceameaningfultargetprofile92,sotherewasobviousroomandneedforabetterworkflow.Usingthedasatinibcapturecompoundpre-clickedtothecopper-chelatingbiotinallowedforthesuccessfulidentificationofbonafidedasatinibtargets inaBCR-ABL-positivecell lysate,suchasABL1,ABL2,BCRandSRC,aswellasotherkinases(Table 3–2 and Figure 3-5, on page 61). Even though the new clickworkflowdid not produce thesame resultsas the full compound in lysate, theseproteinswereenrichedwhencomparing to thenegative controlbutwerenot competedenoughby freedasatinib topass the inclusion criteriaoftwo-times fold-change and p value less than 0.05. Other known dasatinib inhibited kinases weresuccessfullyidentifiedusingthisprotocol.

Searching for dasatinib targets in living cells showed that a large number of biological andtechnical replicates is necessary to achieve statistical significance when probing living cells.Individually, each experiment consisting of technical replicates yielded very different resultsregarding potential targets (Table 3–7, on page 70). Calculating the intensity differences betweenassays and competitions of all experiments as one or attributing a re-incidence score allowed abetter understanding of the results. In fact, using this strategy, known dasatinib targets were


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competitively identified for the first time in living cells,with special relevance to the C-Src kinase(CSK),aswellasthebonafidedasatinib-binderABL1/ABL2.

Havingsuccessfullyimplementedthecaptureofsmallmoleculetargetsinlivingcells,thenewmethodologywasusedtoidentifynewbindersforphenobarbital,adrugthathasbeeninusefor100years and still has unknown mechanisms of action regarding its toxicity. The hepatocarcinogeniceffectofphenobarbitalinsomespecieswasthefocusofthisendeavor,andatthetimeexperimentswere being performed it was not known that EGFR could bind phenobarbital and elicit a cellularresponse,aspublishedbyMutohetal.in2013142.

Inordertolaterbeabletoassociatecaptureresultstophenotypiceffects,thefirstobjectivewas to implement a phenotypic assay that would serve as a validation method once thephenobarbital targetcandidatewas identifiedbymassspectrometry.Several systemswere tested,such as AMPK phosphorylation or CAR translocation that according to literature happens uponphenobarbitalstimulation,buttheresultsobtaineddidnotconfirmthatandcouldthereforenotbeused as reliable readouts. The constitutive androstane receptor (CAR) was already found in thenucleusofunstimulatedcellsandAMPKphosphorylationwascyclicmakingthempoorcandidatesasavalidationmethod.ThesuccessfulphenotypicassayturnedouttobetheexpressionofcytochromeP4502B1mRNAthatclearlyincreasesuponhighdosageofphenobarbital(Figure4-18,onpage96)in both rat hepatoma cell line and primary hepatocytes. Unfortunately, by the time massspectrometryresults indicatedpotential targets tobevalidated, therewasno furtherpossibility tocontinuetheresearchduetotimeconstraints.

The mass spectrometry analysis of the initial capture experiments using a pre-clickedphenobarbitalcapturecompoundinlysatefromarathepatomacelllineprovidedinterestingresults.Mostoftheputativehitshadbeenpreviouslyassociatedtosomephenobarbitaleffectandsomehadalso known interactions with EGFR. In those experiments, subunits from the phosphatase PP2Aresponsible for the de-phosphorylation of AMPK and RACK1, the EGFR-associated kinase that aidsthetranslocationofCARtothenucleus,wereidentifiedascandidatebindersofphenobarbital.Evenmore interestingly,Wdr1 and Ybx1were also presented as putative targets of phenobarbital. Thefirst being a WD repeat-containing protein, characteristic shared with RACK1, and the second aproteinthatbindsY-boxes,specificsequencesingenepromoters,thatarepartofthephenobarbitalresponseenhancermodule(PBREM)presentinCYP2Bgenes.

Results fromcaptureexperimentswithphenobarbitalprobes in livingMH1C1 cells continuedpointing towards a connection to the EGFR signaling pathway. Different proteins were identifieddepending on whether the capture compound was pre-clicked or not, but all have documentedassociationwithEGFR:athioesterase(Them6)andannexinA4(Anxa4)were identifiedaspotentialtargets insamplesprobedwithacompletephenobarbitalcapturecompound;andADP-ribosylationfactor 5 (Arf5) and dynamin-2 (Dnm-2) were found as binders in samples with the clickablephenobarbitalcapturecompound.

All the identifications in the phenobarbital experiments directed the attention for EGFR butthisproteinwasneverspecificallycaptured.Toanswertheposingquestionofwhetherthereceptorwas not targeted because it is not a direct binder of phenobarbital or if it was only due to lowabundanceinthesample,alastexperimentwasperformedinatissuethatexpresseshighlevelsofEGFR.Theresults fromthecapture inhumanplacenta lysateconfirmedsomepreviously identifiedproteinsasputativetargetsofphenobarbitalanditsconnectiontotheEGFRsignalingpathway,but


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thereceptoritselfwasnotcompetedbyphenobarbital.ArecurrentcandidateastheprimarybinderofphenobarbitalwasYBX1,andgivenitsroleastranscriptionfactoranddirectbinderofEGFR,thepleiotropiceffectsand themodulationof theEGFRsignalingcascade in response tophenobarbitaltreatment could be explained. Further validation is necessary to confirm that YBX1 is inhibited byphenobarbital.

Apointof concern, inhindsight, is that thephenotypic effect elicitedbyphenobarbital onlyoccurs at high compound concentrations, close to the millimolar range, suggesting low affinitytowards the pathway under study. To further pursue CCMS in living cellswith permeable capturecompounds,thefocusshouldbeonmorepotentsmallmolecules,elicitingeffectsinthenanomolarrange.

Amongstthedrugtargetdeconvolutiontechniques,CCMSremainsthemostrobustandwithhigherpotentialtoinnovate.Atthetime,probinglivingcellswithbio-orthogonalcapturecompoundsis a reality but the results are still far from ideal. Effortsmust be put in reducing background ofcaptureandmassspectrometrymethodsaswellasexperimentingwithnewclickablepairsthatallowforabetterreactionyieldand,consequently,betteridentifications.

Inmyopinion, thebest strategypresently fordrug targetdeconvolution is to firstly identifywhich subcellular structures contain the target, using bio-orthogonal reactions and microscopyanalysis,fractionateandenrichthoseorganellesandthenuseCCMSinthosesubproteomes.

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6. GeneralMethodsandMaterials6.1. CELLCULTURE6.1.1. HEK293cellculture

Humanembryonickidney(HEK)cells,clone293,wereusedincAMP/PKAcaptureexperiments(asdescribedin2.Labelingaproteinusingclickchemistry,onpage28).

HEK293cellswereculturedinDulbecco’sModifiedEagleMedium(DMEM)supplementedwith10% heat-inactivated fetal bovine serum (FBS) and 1% of penicillin/streptomycin in a 5% CO2incubatorat37°C.Thecellsweresubculturedata1:5–1:6dilutionevery2-3daysbymechanicaldetachment.

6.1.1.1. HEK293lysatepreparation

HEK293 cell pellets were lysed in 2X their volume with a hypotonic buffer (10 mM HEPESpH7.9, 1.5 mM MgCl2, 10 mM KCl) containing or not 0.5% (w/v) DDM. Both buffers weresupplementedwithproteinaseinhibitorsandbenzonaseatthetimeoflysis.After30minincubationon ice, the lysatewasdounced20Xusinga tightpestleandthenrunthrougha21Gneedlewithasyringe.Remainingdebriswaspelletedbyrefrigeratedcentrifugationat15.000rpmfor30min.Thesupernatant was recovered and the leftover pellet was discarded. Aliquots weremade and snap-frozeninliquidnitrogen.Sampleswerestoredat-80°Cuntiluse.

6.1.2. K562cellcultureHumanK562cellsoriginatefromachronicmyeloidleukemiainblastcrisis,andwereusedin

theidentificationofdasatinibtargetsexperiments(asdescribedin3.IdentifyingDasatinibtargetsincells,onpage54).

K562cellswereculturedinRPMI-1640mediumsupplementedwith10%heat-inactivatedfetalbovine serum (FBS) and1%penicillin/streptomycin in a5%CO2 incubator at 37 °C. The cellsweresplitevery3daysata1:3–1:5dilutionbypelletingthesuspensioncellsfor10minat1000xg.

6.1.2.1. K562lysatepreparation

K562cellpelletswerelysedineither(1)RIPAbuffer(50mMTris-HClpH8.0,150mMNaCl,1%IGEPAL®CA-630,0.5%(w/v)sodiumdeoxycholate,0.1%(w/v)SDS)supplementedwith0.2mMDTT;or(2)LysisBufferE(50mMTris-HCl,100mMNaCl,1.5mMMgCl2,0.2%(v/v)IGEPAL®CA-630,0.5%(w/v) DDM, 5% (v/v) glycerol) supplemented with 1 mM DTT. Both buffers were additionallysupplementedwithproteinaseinhibitorsand0.5μL/mlbenzonase.

After30minincubationonice,thelysatewasdounced20Xusingatightpestleandthenrunthrougha21Gneedlewithasyringe.Remainingdebriswaspelletedbyrefrigeratedcentrifugationat15.000 rpm for 30 min. The supernatant was recovered and the leftover pellet was discarded.Aliquotsweremadeandsnap-frozeninliquidnitrogen.Sampleswerestoredat-80°Cuntiluse.

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6.1.3. MH1C1cellculture

AratclonalstrainofepithelialcellsderivedfromMorrishepatoma#7795,MH1C1cellline,wasusedinphenobarbitalexperiments(asdescribedin4.Phenobarbital:newbindersforanolddrug,onpage81).

MH1C1cellswereculturedinDulbecco’sModifiedEagleMedium(DMEM)supplementedwith10%heat-inactivatedfetalbovineserum(FBS)and1%penicillin/streptomycinina5%CO2incubatorat37°C.Thecellsweresplitusingtrypsin/EDTAonceortwiceperweekata1:2–1:4dilution.

6.1.3.1. MH1C1lysatepreparation

MH1C1cellpelletswerelysedinLysisBufferE(50mMTris-HCl,100mMNaCl,1.5mMMgCl2,0.2% (v/v) IGEPAL® CA-630, 0.5% (w/v) DDM, 5% (v/v) glycerol) supplemented with 1 mM DTT,proteinaseinhibitorsand0.5μL/mlbenzonase.

After30minincubationonice,thelysatewasdounced20Xusingatightpestleandthenrunthrougha21Gneedlewithasyringe.Remainingdebriswaspelletedbyrefrigeratedcentrifugationat15.000 rpm for 30 min. The supernatant was recovered and the leftover pellet was discarded.Aliquotsweremadeandsnap-frozeninliquidnitrogen.Sampleswerestoredat-80°Cuntiluse.

6.1.4. RatprimaryhepatocytescultureRat (male Sprague-Dawley) cryopreserved hepatocytes were used in phenobarbital

experiments(asdescribedin4.Phenobarbital:newbindersforanolddrug,onpage81).The cryopreserved hepatocyteswere thawed in a 37 °Cwater bath for less than 2min and

transferredtoapre-warmedtubecontainingWilliamsMediumEandHepatocytePlatingSupplementPack(ThermoFisherScientific).Thecellswerecentrifugedat55xgfor3minatroomtemperature.

Thesupernatantwasdiscardedand1mLper1x106cellsofplatingmediumwasadded.ThehepatocytesweredilutedandseededincollagenI-coated24-wellplatesataconcentrationof8x105cellperwell(500µL).Theplatewasincubatedfor4hat37°Ctoallowadherenceandformationofmonolayer. After incubation the platewas agitated to release unattacheddebris and themediumwasaspiratedandreplacewithfreshcompletemedium.

The ratprimaryhepatocyteswereused forRT-PCRexperiments (asdescribed in4.3Specificmethodsforthephenobarbitalsystem,4.3.6RT-PCR,onpage107).

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6.2. BIOCHEMISTRYGENERALMETHODS6.2.1. Proteinconcentrationdetermination

TotalproteinconcentrationfromcelllysateswasdeterminedusingthecommerciallyavailableBCAProteinAssayReagent(Pierce™,Prod.#23225).TheBCAProteinAssaywasintroducedbySmith,etal.in1985184.Themainbenefitofthisassayisthecompatibilitywithawiderangeofdetergentsnormallyusedincellularlysates.Theassayreliesonacolorimetricdetectionofcopperreductionbybicinchoninicacid(BCA).

A standard curve of bovine serumalbumin (BSA) (Pierce™, Prod #23209)was prepared in adilutionseriesusingthesamelysisbufferofthecellular lysatetobequantified.Thesamplestobequantifiedwerealsodiluted(1:4–1:20)inbuffertoassurethemeasurementiswithintherangeofthestandards.

10μLofBSAstandardorsamplewerepipetted intriplicates intoa96-well flat-bottomplateand200μLofworkingreagent(50:1,reagentA:reagentB)wereaddedtothewells.Theplateswereincubated for 30min at 37°C and the light absorbance read in amultiplate reader (Anthos 2010,AnthosMikrosystemeGmbH)witha562nmfilter.

Thevalueswereinsertedinaspreadsheetandtheaverageofthestandardswasusedtoplotandcalculatealinearregressioncurve.Thetotalproteinconcentrationinthesampleswascalculatedbyinferenceandcorrectedforthedilutionfactor.

6.2.2. SDS-PAGESodiumDodecylSulfatePolyacrylamideGelElectrophoresis(SDS-PAGE)isaproteinanalytical

method established byMaizel 185 in 1963 as an advancement of the original PAGE developed byWeintraubin1959186.ThecurrentmethodmostlyusedaroundtheworldwasoptimizedbyLaemmliin1970187.ForaninsightonthedevelopmentandestablishmentofSDS-PAGEasareferenceproteinanalyticalmethodseeMaizel’sreviewpublishedin2000188.

Proteinsolutions,cellularlysatesorwholecellsweremixedwithLaemmliBuffertoafinal1Xconcentration (50 mM Tris-base, 2.5% (w/v) SDS, 10% Glycerol, 0.32 mM β-mercaptoethanol,bromophenol blue). Samples were loaded on pre-casted 4-20% Tris-Glycine gels (anamedElektrophorese GmbH, Germany) and proteins were separated for 35 min at 300V on a XCellSureLock®Mini-Cell electrophoresis chamber (Life Technologies™GmbH,Germany) filledwith SDSRunningBuffer(25mMTrisbase,192mMGlycine,0.1%(w/v)SDS).Apre-stainedproteinmolecularweightmarkerwasalwaysloadedinonewellasareference.

After separation, gels were directly stained using Coomassie Blue or Silver staining, ortransferredontonitrocellulosemembranesforWesternBlotting.


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6.2.3. PolyacrylamideGelStainingResolvedproteinsonpolyacrylamidegelscanbevisualizedwithspecificdyes.Thetypeofdye

tousewill dependon the abundanceof theproteinsof interest. The first dyeof choice is usuallyCoomassie Brilliant Blue because of its simple protocol and the signal-to-background is, in mostcases, sufficient tomakeanassessmentof theexperiment.More sensitivedyes, suchas theonesusing Silver, usually take longer procedures but are necessary when dealing with low abundantproteins.

6.2.3.1. CoomasieBlue

The Coomassie-based gel stain used to visualize proteins on gels was the SimplyBlue™SafeStain from Invitrogen®. It contains a proprietary Coomassie G-250 stain for a fast sensitivedetectionwithoutrequiringtheuseofmethanoloraceticacid.

The protocolwas followed as described by themanufacturer. After electrophoresis, the gelwasrinsed3timesfor5minwithultrapurewatertoremoveSDSandbuffersalts.Thegelwasthenincubatedforatleast1hwiththecoomassiesolution,gentlyshakingatroomtemperature.Thegelwas thenwashedwithultrapurewater for thenecessary time to reducebackgroundand increasecontrast with stained bands. The gel was kept in water if further mass spectrometry analysis ofindividualbandswasperformed.

6.2.3.2. SilverStain

The ProteoSilver™ Silver Stain Kit (Sigma®) was used to detect low abundant proteins.ProteoSilveruses silvernitrate,whichbinds to selectiveaminoacidson theproteinsunderweaklyacidic or neutral pH conditions. The protein bound silver ions are reduced by formaldehyde atalkalinepHtoformmetallicsilverinthegel.

Theprotocolwasfollowedaccordingtomanufacturer’sguidelines.Thepolyacrylamidegelwasfixed forat least20min in fixingsolution (50%ethanol,10%aceticacid), followedbyawashwith30%ethanolandawashwithultrapurewater,10mineach.Thegelwasthenincubatedfor10mininsensitizer solution (contentsnotdescribed) and thenwashed two times for 10minwithultrapurewater.Afterthat,thegelwasequilibratedinsilversolutionfor10minfollowedbyaquickwashwithultrapurewaterandimmediatelysoakedindevelopersolution(contentsnotdescribed).Incubationtimes in this solutionvarieduntildesiredbandswerevisualized.To stop furtherdevelopmentandincreaseofbackgroundthereactionwasterminatedbyaddingstopsolution(contentsnotdescribed)directly to the immersed gel. The solution was discarded and the gel was washed for 15 min inultrapurewater. The stained gel could be kept in fresh ultrapurewater until further analysiswasnecessary,suchasmassspectrometryanalysisofindividualproteinbands.


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6.2.4. WesternBlottingIn1979,HarryTowbindevelopedamethodtoallowfortheprobingofanyelectrophoretically

separatedproteinwithantibodiesorserum189.Theprinciplebehind itwastocreateacopyoftheelectrophoreticpatternobtainedonthegelbytransferringittoanitrocellulosemembrane189.Twoyears later, Burnette 190 made a few modifications to the transfer buffer conditions to allow acomplete andquantitative elutionof proteins from the SDS gel onto thenitrocellulosemembraneand coined it “Western Blotting”. Thismethod is still today themost commonly used for proteintransfer.

After gel electrophoresis, the resolved proteins were transferred onto a nitrocellulosemembrane (0.2 µm, Whatmann™) using a wet-transfer blotting tank (Biozym™) in either a Tris-glycine homemade transfer buffer or a proprietaryWestern Blot Transfer Buffer from Pierce™. Astack of (bottom-to-top) three layers of Whatmann paper, the nitrocellulose membrane, thepolyacrylamide gel, and another three layers of Whatman paper was assembled in a blottingchamber.All layerswere soakedwithblottingbuffer andelectrotransferwasperformedat 1mA/cm2(45mApergel).Tovisualizeproteins,thenitrocellulosemembranewasstainedwithPonceauSsolution.ThestainwasremovedwithTBS-Twashingandblockedin2%(w/v)non-fatmilk inTBS-Tforatleast1hatroomtemperatureorover-nightat4°C.

For detection of biotinylated proteins, the membrane was incubated with infrared (IR) orhorseradishperoxidase (HRP)-conjugatedstreptavidindiluted1:1000 inblockingsolutionfor2hatroomtemperatureorover-nightat4°C.BlotswerethenwashedthreetimeswithTBS-Tfor10minandkeptinTBSuntilreadout.

Forspecificproteinsdetection,themembranewaswashedthreetimeswithTBS-Tfor10minand then incubated with primary antibody solution (diluted in blocking solution) for 2 h at roomtemperatureorover-nightat4°C.Afterthree10minwashesinTBS-T,themembranewasexposedtoIR or HRP-conjugated secondary antibody solution (diluted in blocking solution) for 1 h at roomtemperature. Finally, the blot was washed three times in TBS-T for 10min and kept in TBS untilimageacquisition.

IR-labeledblotsweredirectlyacquiredintheimagingsystem(SyngeneG:BOXXT4)usingtheappropriatecombinationofexcitationlightandcutofffilters.

For HRP-labeled blots, the membranes were incubated with enhancedchemiluminescence(ECL)detectionreagent(Pierce®)andtheemittinglightwasrecordedintheimagingsysteminthedarkwithoutanyfilter.

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6.3. MASSSPECTROMETRY6.3.1. Sampleacquisitionanddatabasematchidentification

TrypticdigestswereanalyzedbyonlinenanoflowliquidchromatographytandemMS(LC-MSn)onanUltiMate3000RSLCnanoSystem(Dionex,partofThermoFisherScientific,Germany)coupledto a LTQ-Orbitrap Velos instrument (Thermo Fisher Scientific, Germany) through a Proxeonnanoelectrospray ion source (Proxeon, part of Thermo Fisher Scientific, Germany). Forchromatographic separation, samples were first loaded on a reversed phase (RP) pre-column(AcclaimPepMap100,5mm,100Å,100µmi.d.x20mm)andseparatedonaRPanalyticalcolumn(Acclaim PepMap RSLC C18, 2 µm, 100 Å, 75 µm i.d. x 150 mm, Dionex, part of Thermo FisherScientific,Germany)performinga96mingradient(5–45%acetonitrile,0.1%formicacid).

MS detectionwas performed in the data-dependentmode allowing to automatically switchbetweenOrbitrap-MSandLTQ-MS/MSacquisitioninatop20configurationat60Kresolutionforafull scanwith subsequent collision induced dissociation (CID) fragmentation. Full scanMS spectra(fromm/z300–2000)wereacquiredintheOrbitrapanalyzerafteraccumulationtoatargetvalueof1x106inthelineariontrap.Themostintenseions(uptotwenty,dependingonsignalintensity)withchargestate≥2weresequentiallyisolatedatatargetvalueof5000andfragmentedinthelineariontrapusinglowenergyCIDwithnormalizedcollisionenergyof35%.TargetionsalreadymassselectedforCIDweredynamicallyexcludedforthedurationof60s.TheminimalsignalrequiredforMS2was1000 counts. An activation q of 0.25 and an activation time of 10 ms were applied for MS2acquisitions.

AllMS/MSdatawereanalyzedusingAndromeda implemented inMaxQuant 120. Automateddatabase searching against the humanUniProtKB/Swiss-Prot databasewas performedwith 6 ppmprecursortolerance,0.5Dafragmentiontolerance,fulltrypsinspecificityallowingforupto2missedcleavages and methionine oxidation as variable modification. The maximum false discovery ratesweresetto0.01bothonproteinandpeptidelevel,themaximumPEPto1,and7aminoacidswererequired as minimum peptide length. The label free quantification option was selected with amaximalretentiontimewindowof2minforthealignmentbetweenLC-MS/MSruns.

Calculations for fold-changebetweendifferentsubsetsof samplesandStudent’s t-TestwereperformedmanuallyinExcelspreadsheetsusingbothMS-basedvalues(LFQvaluesfromMaxQuant)andalignment-basedintensityvalues.

CCMSchartsweredevelopedtocomprehensivelydisplaythemulti-parameterresultsobtainedinCCMSexperiments for individualproteins,plottingminimumandmaximumintensityvaluesasaboxwithablackhorizontalbar for themedianvalue± thestandarderrorof themean (SEM). Inaseparate axis, the fold-change between the subsets of samples is plotted as a green triangle andsignificant(p<0.05)differencesarehighlightedwithanasterisk(*).

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6.4. MOLECULARSTRUCTURESOFCOMPOUNDS6.4.1. CapturecompoundsCC1:ClassicalfullcAMPcapturecompound

CC2:ClickablecAMPalkynecapturecompound(containsselectivityandphotoreactivefunctions)

CC3:Clickablebiotin-PEG-azide(containslabelingfunction)

CC4:ClickedcAMP-biotincapturecompound.ReactionproductofCC2andCC3


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CC5:ClickableTAMRA[tetramethylrhodamine]azide(containslabelingfunction)

CC6:ClickableDasatinibalkynecapturecompound(containsselectivityandphotoreactivefunctions)


CC8:ClickableTAMRA-PEGcopper-chelatingazide(containslabelingandcopperligandfunctions)


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CC9:Clickeddasatinib-biotincapturecompound.ReactionproductofCC6andCC7

CC10:Clickablephenobarbitalalkynecapture

compound A (contains selectivity and a

trifluoromethyl-phenyl-diazirine photoreactive

functions)

CC11:Clickablephenobarbitalalkynecapture

compound B (contains selectivity and a

methyl-diazirinephotoreactivefunctions)

CC 12: Clickable phenobarbital alkyne capture compound C (contains selectivity and a azido-

tetrafluorobenzenephotoreactivefunctions)


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6.4.2. CompetitorsCC13:cAMPcompetitor

CC14:Dasatinibcompetitor

CC15:Phenobarbitalcompetitor

6.4.3. LigandsCC16:

Copper-chelatingligandTBTA

Tris(benzyltriazolylmethyl)

amine

CC17:

Copper-chelatingligandTHPTA

Tris(3-hydroxypropyltriazolylmethyl)

amine

CC18:

Copper-chelatingligandBTTE

bis(tert-butyltriazoly)ethanol

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6.5. MATERIALS6.5.1. Chemicalreagents

ChemicalnameMolecularFormula

Molarmass(g/mol)

CAS SupplierRiskandSafetyStatements

(+)-SodiumL-ascorbate C6H7NaO6 198.11 134-03-2 Sigma -

Acetonitrile(HPLCgrade) C2H3N 42.05 75-05-8 SigmaR:11-20/21/22-36S:16-36/37

Ammoniumbicarbonate NH4HCO3 79.06 1066-33-7 Sigma R:22

Biotin C10H16N2O3S 244.31 58-85-5 BioMag -

BromophenolBluesodiumsalt

C19H9Br4NaO5S 692.00 34725-61-6 Serva S:22-24/25

Copper(II)sulfatepentahydrate

CuSO4•5H2O 249.69 7758-99-8AlphaCaesar

R:22-36/38-50/53S:22-60-61

Dimethylsulfoxide C2H6SO 78.13 67-68-5 Acros -

DL-Dithiothreitol C4H10O2S2 154.25 3483-12-3 FlukaR:22-36/37/38S:26-36

EDTA-Na2 C10H14N2Na2O8•2H2O 372.24 6381-92-6 Gerbu -

Formicacid CH2O2 46.03 64-18-6 FlukaR:35S:23-26-45

Glycerol C3H8O3 92.09 56-81-5 Gerbu -

Glycine C2H5NO2 75.07 56-40-6 Gerbu S:22-24/25

Hepes C8H18N2O4S 238.30 7365-45-9 Gerbu -

Hydrochloricacid HCl 36.46 7647-01-0 SigmaR:34-37S:26-45

IGEPAL®CA-630 (C2H4O)nC14H22O - 9036-19-5 FlukaR:41-50S:26-39-61

Iodoacetamide C2H14INO 184.96 144-48-9 ApplichemR:25-42/43-53S:22-36/37-45

Lutensol®GD70 - - 31799-71-0 BASFR:41S:39-26

Magnesiumacetatetetrahydrate

C4H14MgO8•4H2O 214.45 16674-78-5 Sigma -

Potassiumacetate C2H3KO2 98.14 127-08-2 Sigma -

Potassiumhydroxide KOH 56.11 1310-58-3 SigmaR:22-35S:26-36/37/39-45

Sodiumchloride NaCl 58.44 7647-14-5 Roth -

Sodiumdeoxycholate C24H39NaO4 414.55 302-95-4 Sigma R:22-37

Sodiumdodecylsulfate C12H25NaO4S 288.38 151-21-3 GerbuR:11-21/22-36/37/38S:26-36/37

Sodiumhydroxide NaOH 40.00 1310-73-2 CarlRothR:35S:26-37/39-45

Trifluoroaceticacid(99%) C2HF3O2 114.02 76-05-1 AcrosR:20-35-52/53S:9-26-27-28-45-61

Tris(hydroxymethyl)aminomethane

C4H11NO3 121.14 77-86-1 VWR R:36/38

Triton™X-100C14H22O(C2H4O)n,n=9-10

x̄=625 9002-93-1 FlukaR:22-36-51/53S:26-39-61

Trypanblue C34H24N6Na4O14S4 960.82 72-57-1 Fluka R:45S:53,45


127

ChemicalnameMolecularFormula

Molarmass(g/mol)

CAS SupplierRiskandSafetyStatements

Trypsin(sequencinggrade,modified,porcine)

- - 9002-07-7 PromegaR:36/37/38-42-42/43S:22-24-26-36/37-45-23

Tween®20 - 1228 9005-64-5 Sigma -

Β-Mercaptoethanol C2H6OS 78.13 60-24-2 ApplichemR:23/24/25-38-41-43-48/22-50/53S:26-36/37/39-45-60-61

N-Dodecylβ-D-maltoside C24H46O11 510.62 69227-93-6 Sigma -

6.5.2. KitsandConsumablesCategory Product ManufacturerAntibody PKARIα BDBiosciencesAntibody AMPKα1/2(H-300) SantaCruzBiotechnologyAntibody CAR1/2(M-127) SantaCruzBiotechnologyAntibody p-AMPKα1/2(Thr172) SantaCruzBiotechnologyAntibody CYP2B1/2B2(9.14) SantaCruzBiotechnologyBiotindetection HRP-conjugatedStreptavidin SigmaBlottingBuffer 10XWesternBlotTransfer

Buffer,Methanol-freePierce,Thermo

CellFractionation NE-PER®NuclearandCytoplasmicExtractionReagents

PierceBiotechnology

GelStaining SimplyBlue™SafeStain InvitrogenGelStaining ProteoSilver™SilverStainKit SigmaHorseradishperoxidasedetection

SuperSignalWestDuraChemiluminescentSubstrate

Pierce,Thermo

MagneticBeads DynaBeads MembraneStain Ponceau Molecularweightmarker PageRulerPlusPrestained

ProteinLadderPierce,Thermo

PolyacrylamideGels 4-20%Tris-GlycinGel AnamedElektrophoreseGmbHProteaseinhibitor cOmplete,Mini,EDTA-free

ProteaseInhibitorCocktailTablets

Roche

ProteinQuantificationAssay BCAProteinAssayReagent Pierce,Thermo


128

6.5.3. Buffers,mediaandsolutions StockSolution WorkSolutionSDSElectrophoresisRunningBuffer 10x 250mMTris

1.92MGlycine1%(w/v)SDS

1x For1L:100mLstock+900mLH2O

BlottingBuffer 10x 478mMTris386mMGlycine0.37%(w/v)SDS

1x For1L:100mLstock+700mLH2O+200mLMethanol

TrisBufferedSaline(TBS) 10x 0,5MTris1,5MNaClpH7.5(withHCl)

1x For1L:100mLstock+900mLH2O

LaemmliBuffer 4x 200mMTris-BasepH6.810%(w/v)SDS40%(v/v)Glycerol1.28Mβ-MeBromophenolblue

2x For1mL:500μLstock+500μLH2O

Radioimmunoprecipitationassay(RIPA)buffer

1x 150mMNaCl1%(v/v)IGEPAL®CA-6300.5%(w/v)sodiumdeoxycholate0.1%(w/v)SDS50mMTrispH8.0

Usestock

WashingBuffer 5x 250mMTris5mMEDTA-Na25MNaCl0.25%(v/v)LutensolGD70pH7.5(withHCl)


CaptureBuffer 5x 100mMHEPES250mMPotassiumacetate50mMMagnesiumacetatetetrahydratepH7.9(with1MKOH)50%(v/v)Glycerol


LysisBufferE 1x 50mMTris-HCl100mMNaCl1.5mMMgCl20.2%(v/v)IGEPAL®CA-6300.5%(w/v)DDM5%(v/v)Glycerol1mMDTT

Usestock

CellCultureMediaandSupplements Cellline RPMI1640 K562;HEK293 WilliamsMediumE Primaryhepatocytes DMEM MH1C1 Heatinactivatedfetalbovineserum(FBS) - Penicillin/Streptomycinsolution -


129

6.5.4. Equipment

Category Model/type ManufacturerCaprobox 310and350nm caprotecbioanalyticsGmbH,Berlin,DECentrifuge Microstar17R VWRInternationalGmbH,Darmstadt,DECentrifuge 5702R EppendorfAG,Hamburg,DECentrifuge MicrocentrifugeAL CarlRothGmbH,Karlsruhe,DECentrifuge MinicentrifugeMCF-2360 LMSConsultGmbH,Brigachtal,DEElectrophoresis BlottingsystemEBX-700 C.B.S.Scientific,SanDiego,CA,USA

Electrophoresis NovexMini-CellLife Technologies, Thermo Fischer ScientificGmbH,Dreieich,DE

Electrophoresis PowerSupply300V VWRInternationalGmbH,Darmstadt,DEIceMachine ZNE125 ZiegraEismachinenGmbH,Isernhagen,DE

ImagingSystem SyngeneG:BOXXT4Integrated Scientific Solutions Inc., San Diego,CA,USA

Incubator HeraCell150 ThermoFischerScientificGmbH,Dreieich,DEMagneticStirer RCTbasic IKA-WerkeGmbH,Staufen,DEMassSpectrometer LTQVelos ThermoFischerScientificGmbH,Dreieich,DEMassSpectrometer LTQXL ThermoFischerScientificGmbH,Dreieich,DEMassSpectrometer amaZonspeed BrukerCorporation,Billerica,MA,USA

Microscope InvertedMicroscopeDMILLeica Mikrosysteme Vertrieb GmbH, Wetzlar,DE

nHPLC Ultimate3000Dionex, Thermo Fischer Scientific GmbH,Dreieich,DE

pHmeter SevenEasyS20 MettlerToledoGmbH,Gießen,DEPipetting FinnpipetteNovusiMCP8 ThermoFischerScientificGmbH,Dreieich,DEPipetting ManualResearch EppendorfAG,Hamburg,DERotator Multimix115V VWRInternationalGmbH,Darmstadt,DEShaker RockingShakerRocky LabortechnikFröbelGmbH,Lindau,DEShaker OrbitalShakerOS-10 BioSan,Riga,LVSonicator Sonorex Bandelin,Berlin,DE

SpectrophotometerMicroplatereaderAnthos2010

anthosMikrosystemeGmbH,Krefeld,DE

Spectrophotometer VarianCary50Bio AgilentTechnologies,Inc.,SantaClara,CA,USASpeedVac EZ-2plus GeneVacInc,StoneRidge,NY,USAThermocycler TGradient BiometraGmbH,Göttingen,GermanyThermo-Shaker TS-100 BioSan,Riga,LVVortex Genie2 ScientificIndustries,Inc.,Bohemia,NY,USAWeighingscales SI-234A DenverInstrument,Bohemia,NY,USAWeighingscales XP105DeltaRange MettlerToledoGmbH,Gießen,DE

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