bcr‐abl and gleevac exercise
Post on 23-Dec-2021
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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
Ver.6‐D.SinhaandL.Alemán
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
Ver.6‐D.SinhaandL.Alemán
PROTEINSTRUCTUREBASICSEachproteinhasthefollowingthreelevelsofproteinstructure:PrimarystructureListstheaminoacidsthatmakeupaprotein’ssequence,butdoesnotdescribeitsshape.SecondarystructureDescribesregionsoflocalfoldingthatformaspecificshape,likeahelix,asheet,oracoil.TertiarystructureDescribestheentirefoldedshapeofawholeproteinchain.Inaddition,someproteinsinteractwiththemselvesorwithotherproteinstoformlargerproteinstructures.HowtheseproteinsinteractandfoldtoformalargerproteincomplexistermedQuaternarystructure.
CHEMICALSTRUCTURESOFTHEAMINOACIDSThe20aminoacidsshareacommonbackboneandaredistinguishedbydifferent‘R’groups,highlightedinvariouscolorsbelow.
Name________________________
StarBiochem
Ver.6‐D.SinhaandL.Alemán
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
Ver.6‐D.SinhaandL.Alemán
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)
#271
Name________________________
StarBiochem
Ver.6‐D.SinhaandL.Alemán
#286
#290
#315
#318
#381
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
Ver.6‐D.SinhaandL.Alemán
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
Ver.6‐D.SinhaandL.Alemán
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.
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