hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons...
Transcript of hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons...
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h"p://www.molso,.com/images/icm/image036.jpg
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“Tounderstandasystem,youneedtoperturbit.”
MoleculargeneCcstechniquesMutaConsKnock‐outsKnock‐insRNAiSmallmolecules
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ChemicalgeneCcs
TheuseofsmallmoleculestomodulatespecificfuncConsofaprotein
h"p://en.wikipedia.org/wiki/Glivec
Ex:ImaCnib(Gleevec)
InhibitsreceptortyrosinekinaseacCvityofbcr‐ablfusiongene
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AdvantagesofchemicalgeneCcs
PerturbspecificfuncConsoftargetprotein
Temporalcontrol
Reversible
Drugdevelopment
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Twomethodsofsmallmoleculescreening
ExperimentalscreeningBindingandphenotypeassaysTime‐consuming,expensive
Structure‐basedvirtualscreeningComputerprogramsesCmatebindingmatchesLowsuccessrate
BothrequirecharacterizaConofproteinclasses
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Chemicallibraries
Diversity‐orientedlibrariesMoleculesdonotcloselyresembleeachother
FocusedlibrariesSimilarcoretomolecules
TwoapproachesarecomplementaryStockwell.Nature432,846‐854,2004
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Howdowedefinediversity?
Differencesinchemicalstructure
DifferencesinbiologicalacCvity
WhichismoreusefulforchemicalgeneCcsstudies?
Stockwell.Nature432,846‐854,2004
Hu,et al.Nature Reviews Cancer 7,23‐24,2007
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Methodsofexperimentalscreening
Protein‐bindingassaysProtein+moleculeinteracConbiologicalacCvity
“ReversechemicalgeneCcs”
PhenotypicacCvityassaysMolecule+organismphenotypeprotein
“ForwardchemicalgeneCcs”
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Protein‐bindingassays
LabeledFluorescenceorradioacCvityHighthroughputTimeconsumingLabelinterference
UnlabeledLowthroughputMoreprotein/moleculetouseCosteffecCve
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Labeledprotein‐bindingassays
Smallmoleculemicroarray
Findtargetmoleculeforaspecificprotein
Testthousandsofcompounds
Stockwell.Nature432,846‐854,2004
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Labeledprotein‐bindingassays
Proteinmicroarray
Findwhichproteinsamoleculewillbind
Stockwell.Nature432,846‐854,2004
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Labeledprotein‐bindingassays
DNAexpressionplasmidarrays
Chiphasplasmidsthataretakenupbyplatedcells
Testsbindingofmoleculeinalivecell(physiological)
Stockwell.Nature432,846‐854,2004
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Labeledprotein‐bindingassays
Threehybridsystem
Bindingofcompoundtoproteinallowsexpressionofareportergene
Stockwell.Nature432,846‐854,2004
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Unlabeledprotein‐bindingassays
Surfaceplasmonresonance
Similartosmallmoleculemicroarray,butmeasurerefracCvityofsurface
Stockwell.Nature432,846‐854,2004
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PhenotypicacCvity
Characterizedbasedon:
Cellularphenotype
Organismalphenotype
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PhenotypicacCvityassays
Lowthroughput*Mostmustbecalculatedbyhand*ThereareexcepCons
MicroarraysalsousedExaminephenotypicspecificgeneexpression
Stockwell.Nature432,846‐854,2004
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PhenotypeacCvityassays
ImagingAutomatedmicroscopes
RNAandproteinexpressionMaybeadvantageousoverDNAmicroarraydataforspecificproteins
Tendstobeashi,towardslessbiasedtechniques
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Lackofspecificity—problems
Toxicity
Confoundingresults
Unexpectedresults
Dose‐dependenteffects
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Lackofspecificity—soluCons
Useofpotentchemicals
Runlargescaleprotein‐bindingassays
ComplementresultswithRNAitechniques
Useseveralsmallmoleculeswithredundancy
StudyeachmoleculeatarangeofconcentraCons
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Goal:UsesmallmoleculeandRNAitechniquestoidenCfyproteinsimportantincytokinesisusingDrosophila
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Screens
RNAi19,470dsRNAsscreenedintriplicate
Smallmolecule51,000moleculesscreened(intriplicate?)
QuanCficaConbyautomatedfluorescencemicroscopyforbinucleatecellsDouble‐checkedvisually
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Smallmoleculescreen
25compoundsidenCfiedasmostpotentBinucleines1‐25
TestedagainatvaryingconcentraConsIniCalscreenat25μM
100μM30μM10μM
Eggert,et al.PLoS Biology 2(12),2135‐43,2004
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Smallmoleculescreen
Alsotestedbinucleines1‐25inothercellsHeLa 16/25(64%)BSC‐1 13/25(52%)S. cerevisiae 12/25(48%)
TestedeffectsonacCnpolymerizaConInhibiConbybinucleines4,6,24,and25Binucleines24and25werecontrolsknowntodisruptmicrofilamentpolymerizaCon
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RNAiscreen214genesfoundEffectswereweakeroverallthanthesmallmolecules
Onenewgenefound
Eggert,et al.PLoS Biology 2(12),2135‐43,2004
Cytokinesis
Nucleicacidsynthesisanddegrada;on
Uncharacterized
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Comparingphenotypes
RNAi Smallmolecule
Eggert,et al.PLoS Biology 2(12),2135‐43,2004
b=binucleatelc=binucleatewithlowcellcountd=binucleatewithlarge,diffuseDNAMT=binucleatewithmicrotubuleextenCons
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Comparingphenotypes
A:binucleateB:binucleatewithlarge,diffuseDNAC:binucleatewithlowcellcountD:binucleatewithmicrotubuleextensions
RNAi
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Comparingphenotypes
SmallmoleculeAddiConalphenotypes,includingappearanceofcleavagefurrow
Timing
GainoffuncCon
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Pathwaystargetedbysmallmolecules
AcCncortexintegrityAssociatedwithmicrotubuleextensionphenotype
AuroraBpathwayAssociatedwithlarge,diffuseDNAphenotypeMitoCcdefect
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AuroraBpathway
RNAiidenCfiedAuroraB,INCENP,andSurvivininvolvementinphosphorylaCnghistoneH3andchromosomea"achmenterrorinmitosis
Foundinthechromosomalpassengercomplex
ThenewgeneidenCfiedbyRNAiscreenlocalizestothechromosomepassengercomplexCG4454,orBorealin‐related(Borr)SequencesimilaritytohumanBorealin
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AuroraBpathway
Arrowsindicateabsenceofphospho‐histoneH3
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Summary
StrongerphenotypesidenCfiedwithsmallmolecules
PhenotypeswereverysimilarbetweensmallmoleculeandRNAiscreens
CouldidenCfypathwaysdisturbedbysmallmoleculesbycomparingtoknownRNAitargetphenotypes
CouldnotdirectlyidenCfyspecifictargetsofsmallmoleculeswithmethodsused
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WherechemicalgeneCcshasgo"enus
TaxolScreenforanC‐canceracCvity
Bindsandstabilizesmicrotubules,prevenCngcelldivision
AstemizoleOriginallyusedasananC‐histamine
Foundtoinhibitmalaria‐causingmicrobe
h"p://en.wikipedia.org/wiki/File:Taxol(Paclitaxel)3D.png
h"p://en.wikipedia.org/wiki/File:Astemizole.svg
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Websitestocheckout
h"p://www.hhmi.org/biointeracCve/index.htmlHowardHughesMedicalInsCtuteBiointeracCve
GreatvideosandanimaCons
h"p://www.drugbank.ca/search/seqqueryDrugBanksequencequery
Searchdrugsknowntobindtoyourproteinofinterest
h"p://pubchem.ncbi.nlm.nih.gov/PubChemcompoundsearchBestforfindingmoreinformaConaboutknowncompounds
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DrugBank
Inputsequencehere—nucleoCdeorprotein
Changematrix…PAMandBLOSUMused
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DrugBank
Easytofindreferences!!
Resultsshouldshowuphere,butnoluckformyproteinyet
Reminderofwhichmatrixyouchosetouse
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PubChem
Entryfoundbysearching“imaCnib”—searchesforbcr‐ablgivesonlyoneresult,however,andwasnotthisentry
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StudentquesCons
ThereviewsuggeststhatchemicalgeneCcscanhaveadvantagesovermutaConalapproachesinthatwithmutaConsyoulooseallfuncConsofaprotein,whilewithchemicalgeneCcsyoucana"ackspecificfuncCons.Isthisalwaystrue?Wouldn'titbepossibletoknock‐outonlyaspecificfuncConthroughselecCvedeleCons,orsinglebasepairmutaCons?
Notalwaystrue:canintroducemissensemutaConsinanacCvesiteforexample
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StudentquesCons
IntheEggertpaper,theytalkedabouthowtheconcentraConandCmingofthetreatment(whetherRNAiorsmallmolecule)canaffecttheoutcome,specificallywithrespecttotheAuroraBpathway.HasthisinformaConhelpedinunderstandingtheclinicaltrialresultsfromcancercellstreatedwithAurorainhibitorsastheypredicted?
Thismaybeagoodtheory,butgenerallyifatreatmenthasharmfuleffectsinaclinicaltrial,itiso,enabandonedaltogether.
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StudentquesCons
TheyalsomenConthattheythinkanaturalproductextractfromCowaniamexicanacausedagain‐of‐funcConeffectbecausenodsRNAcausedthesamephenotype.Doyouthinkthisisareasonableconclusionbasedonthis,inlightofthevariabilityinRNAiresultswehaveseenandthefactthatitisthoughtthatsuppressionofagenebyRNAimayresultinanincreaseinexpressionofothergenes?
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StudentquesCons
Seeingashowthecellisacomplexanddynamicenvironment,itwouldseemtometobeimpossibletoknowifthemoleculethatyouthinkyouareusing(fromyourchemicalgeneCclibrary),isactuallywhatyouareusing.Couldn'titbemodifiedbythecellanditscomponentswithoutusknowingit?
Yes!!SomedrugswetakemustbemetabolizedinourbodiesbeforetheyareactuallyacCve.
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StudentquesCons
CanchemicalgeneCctechniquesbeespeciallyusefultoepigeneCcstudiessincemajorepigeneCcregulatorymethodsincludemethylaCon,acteylaCon,andotherchemicaladdiConstoDNA?
OneexampleofstudyofchemicalgeneCcsinepigeneCcs:genisteinfoundinsoyproductsisassociatedwithlowincidenceofcancers