BCR‐ABL and Gleevac Exercise

Name________________________

StarBiochem

Ver.6‐D.SinhaandL.Alemán

BCR‐ABLandGleevacExerciseLearningObjectivesInthisexercise,youwilluseStarBiochem,aprotein3D‐viewer,toexplore:• theinteractionoftheABLtyrosinekinasewithGleevac,asmallmoleculeABLinhibitor• pointmutationsintheBCR‐ABLproteinthatmayconferdrugresistancetoGleevac

BackgroundTheABLproto‐oncogeneencodesacytoplasmicandnuclearproteintyrosinekinasethathasbeenimplicatedinprocessesofcelldifferentiation,celldivision,celladhesion,andstressresponse.Chronicmyelogenousleukemia(CML)iscausedbyareciprocaltranslocationbetweenchromosomes9(containingtheABLgene)andchromosome22(containingtheBCRgene).Thisreciprocaltranslocationresultsinalongerchromosome9,ashorterchromosome22(calledPhiladelphiachromosome)andthefusionoftheBCRandABLgenes.FusionoftheBCRandABLgenesinhematopoieticstemcellsleadstoCML.InwildtypehematopoeiticcellstheABLandBCRgenesproducetwoseparateproteinsthatareonlyactivatedwhentheirsignaltransductionpathwaysareturnedon.Incontrast,thefusedBCR‐ABLgeneencodesasingleproteinthatisactiveirrespectiveofthepresenceofactivatingsignals.ConstitutivelyactivationoftheBCR‐ABLtyrosinekinaseleadstouncontrolledproliferation,reduceddifferentiation,andenhancedsurvivalofhematopoieticstemcells.Theseeffectsareleadingcausesofmanyhematologicalmalignancies.

AformoftheBCR‐ABLfusionprotein,p210,isexpressedin95%ofpatientswithCMLand25‐30%ofpatientswithacutelymphoblasticleukemia(ALL).Anotherformofthisprotein,p190,isexpressedin33%ofpatientswithALL.

Nagar,BetalcancerResearch2002,62,4236‐4243

Name________________________

StarBiochem

Imatinibmesylate(Gleevac,STI571),adrugthattargetsthetyrosinekinaseactivityoftheBCR‐ABLfusionprotein,isaneffectivetreatmentforCMLpatients.ItbindstothekinasedomainoftheBCR‐ABLproteinanddisablesthekinasebypreventingATPfrombindingtotheactivesite.GleevacmaynotworkinadvancedstageCML,becausecancercellsbecomemoregeneticallyunstableandcandeveloppointmutationswhichinterferewithGleevacbinding.ThesepointmutationsarethemajormechanismforthedevelopmentofdrugresistanceinCMLpatientsanddemonstratetheneedtodevelopnewdrugsthatcanavoidresistance.GettingstartedwithStarBiochemTohelpyoulearnhowtousetheprogram,atutorialisavailableunderStarBiochemUserGuide.• TogettoStarBiochem,pleasenavigatetohttp://web.mit.edu/star/biochem.• ClickontheStartbuttontolaunchtheapplication.• ClickTrustwhenapromptappearsaskingyouifyoutrustthecertificate.• InthetopmenuunderFileclickonOpen/Import,select“1fpu”andclickOpen.

YouarenowviewingthestructureofaGleevacvariantboundtotheATPbindingdomainoftheABLprotein,witheachbondintheproteindrawnasaline(“bondsonlyview”).

Practicechangingtheviewpointofthisproteinintheviewwindow. Mac PCTOROTATE

clickanddragthemouse left‐clickanddragthemouse

TOMOVEUP/DOWNRIGHT/LEFT

apple‐clickanddragthemouse

right‐clickanddragthemouse

TOZOOM

option‐clickanddragthemouse

Alt‐left‐clickanddragthemouse

TakeamomenttolookatthestructureofABL(1FPU)boundtotheGleevacvariantfromvariousanglesinthis“bondonly”view.

Name________________________

StarBiochem

PROTEINSTRUCTUREBASICSEachproteinhasthefollowingthreelevelsofproteinstructure:PrimarystructureListstheaminoacidsthatmakeupaprotein’ssequence,butdoesnotdescribeitsshape.SecondarystructureDescribesregionsoflocalfoldingthatformaspecificshape,likeahelix,asheet,oracoil.TertiarystructureDescribestheentirefoldedshapeofawholeproteinchain.Inaddition,someproteinsinteractwiththemselvesorwithotherproteinstoformlargerproteinstructures.HowtheseproteinsinteractandfoldtoformalargerproteincomplexistermedQuaternarystructure.

CHEMICALSTRUCTURESOFTHEAMINOACIDSThe20aminoacidsshareacommonbackboneandaredistinguishedbydifferent‘R’groups,highlightedinvariouscolorsbelow.

Name________________________

StarBiochem

1WewillfirstlookattheoverallstructureofABL.

a)WouldyouclassifytheABLproteinasahomodimeroraheterodimer?• ClickonStructureandselectPrimary.Theaminoacidsofeachpolypeptidechain/monomerarehighlightedbyaspecificcolorandcanbedistinguishedfromthoseofotherpolypeptidechains.

• Comparethesequenceandnumberofaminoacidsbetweenthetwomonomers.• Todistinguishbetweenthedifferentmonomersthatmakeupthisprotein,underStructureclickonQuaternary.

• ClickonChain.Answer

b)DoesthisstructurerepresentthefulllengthABLprotein?AnswerYes/Noandexplainyourchoice.

Answer

2WewillnowstudytheinteractionbetweenGleevacandABL.

a)HowmanyGleevacmoleculesareboundtoeachABLmonomerinthisstructure?Hint:lookatthemoleculesshowninyellow.

Answer

b)DrawthestructureoftheGleevacmoleculepresentinthisstructure.• UnderPDBtree,clickon1fpuandthenclickon“PRC_1”or“PRC_2”.• IntheViewControlspanel,settheUnselectedtransparencysliderto“0”.• ClickonDrawwithintheAtomsboxtoseewhatatomsarepresent.Eachatomiscolorcoded:Carbonisgrey,Nitrogenisblue,OxygenisredandSulfurisorangeinthisexercise.

Answer

Name________________________

StarBiochem

3WewillnowanalyzethesecondarystructureofABL.• WithinthemainmenugotoView.• ClickonResetmolecule.• UnderStructure,clickonSecondary.• ExplorethedifferentsecondarystructuresbyindividuallyclickingHelices,SheetsorCoils.• Alternatively,clickonAllRibbonswithintheShowRibbonsboxtoviewallthesecondarystructuresatthesametime.

a)Howmanystructuresarepresentforeachtypeofsecondarystructure?

Answer

b)Carefullylookattheregionthatbringsthetwomonomerstogether.Whichsecondarystructure(s)interacttobringthetwomonomerstogether?

Answer

c)CarefullyanalyzetheregionoftheproteinthatinteractswithGleevac.Whichsecondarystructure(s)ispresentwithinthisregionoftheprotein?

Answer

4WewillnowcreateastereoviewofthedrugbindingpocketofABL.Gleevacinteractswiththesidechainsofaminoacids271,286,290,315,318and381.

a)Identifytheseaminoacids,drawtheirsidechains,andclassifythem.

Aminoacid# Name Sidechain Acidic,basic,polar,and/ornonpolar(stateallthatapply)

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StarBiochem

b)SketchthedrugbindingpocketofABLboundtoGleevacbydrawingthechemicalstructureofGleevacandthesidechainsofeachoftheseinteractingaminoacids.LabeltheGleevacmoleculeandeachofthesidechainsinvolved.

• UnderStructure,clickonPrimary.• HighlighttheaminoacidsstateaboveforthefirstAblmonomerbyindividuallyclickingonthemandsimultaneouslypressingControlandApplekey(Mac)/right‐click(PC).

• UnderPDBTree,clickonthe1fpufile.Clickon“PRC_1”.• IntheViewControlspanel,settheUnselectedtransparencysliderto“0”whilekeepingtheSelectedtransparencysliderat“1”.

• ClickonDrawintheAtomsboxtoseewhatatomsarepresent.Eachatomiscolorcoded:Carbonisgrey,Nitrogenisblue,OxygenisredandSulfurisorangeinthisstructure.

Answer

Name________________________

StarBiochem

b)StatethemostlikelymodeofinteractionbetweenthesidechainsofeachoftheseaminoacidsandtheGleevacmoleculeinthisstructure.Indicatethegroupsinvolved.Yourchoicesare‘ionic’,‘hydrogen’,‘vanderWaalsforces’,‘hydrophobic’or‘covalent’.

Answer

c)WhichoftheseaminoacidsmayberesponsibleforABL’skinaseactivity?Explainyouranswer.Hint:paycloseattentiontothesidechainsofthespecifiedaminoacidsinpart(a)whileansweringthisquestion.

Answer

d)BasedonwhatyouhavelearntfromQuestion4andtheinformationpresentedinthe‘Background’section,proposeamechanismthatexplainshowGleevacinhibitsABL’skinaseactivity.

Answer

5SpecificpointmutationsintheBCR‐ABLproteinmayconferresistancetoGleevactreatment.Foreachofthefollowingpointmutations,explainhoweachofthesemutationsmightconferresistancetoGleevactreatment.

Answer‐Leu248Val ‐Tyr253His ‐Thr315Ile

Name________________________

StarBiochem

Keywords:Kinase,pointmutations,drugresistance,reciprocaltranslocation,chromosome,Philadelphiachromosome,hematopoeticstemcells,constitutivelyactiveprotein,celldifferentiation,cellproliferation,hematologicalmalignancies,Gleevac,oncogenes,proto‐oncogenes,tumorsuppressorgenes,andcellcycle.Thoughtquestions1“Theoriginandproliferationofacancercellisanexcellentexampleofmicroevolution.”Elaborateandexplainthisstatement.

2TheBCR‐ABLproteinhasthreedifferentisoforms:p210,p190andp230.Proposeatheorytoexplainhowtheseisoforms/variantsofBCR‐ABLcanbeproduced.

3Cellproliferationisregulatedbythefinebalancebetweenthefunctionofproto‐oncogenesandtumorsuppressorgenes.WouldyouclassifythewildtypeABLgeneasanoncogeneoratumorsuppressorgene.Explainyouranswer.

BCR‐ABL and Gleevac Exercise

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