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Page 1: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

h"p://www.molso,.com/images/icm/image036.jpg

Page 2: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

“Tounderstandasystem,youneedtoperturbit.”

MoleculargeneCcstechniquesMutaConsKnock‐outsKnock‐insRNAiSmallmolecules

Page 3: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

ChemicalgeneCcs

TheuseofsmallmoleculestomodulatespecificfuncConsofaprotein

h"p://en.wikipedia.org/wiki/Glivec

Ex:ImaCnib(Gleevec)

InhibitsreceptortyrosinekinaseacCvityofbcr‐ablfusiongene

Page 4: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

AdvantagesofchemicalgeneCcs

PerturbspecificfuncConsoftargetprotein

Temporalcontrol

Reversible

Drugdevelopment

Page 5: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Twomethodsofsmallmoleculescreening

ExperimentalscreeningBindingandphenotypeassaysTime‐consuming,expensive

Structure‐basedvirtualscreeningComputerprogramsesCmatebindingmatchesLowsuccessrate

BothrequirecharacterizaConofproteinclasses

Page 6: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Chemicallibraries

Diversity‐orientedlibrariesMoleculesdonotcloselyresembleeachother

FocusedlibrariesSimilarcoretomolecules

TwoapproachesarecomplementaryStockwell.Nature432,846‐854,2004

Page 7: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Howdowedefinediversity?

Differencesinchemicalstructure

DifferencesinbiologicalacCvity

WhichismoreusefulforchemicalgeneCcsstudies?

Stockwell.Nature432,846‐854,2004

Hu,et al.Nature Reviews Cancer 7,23‐24,2007

Page 8: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Methodsofexperimentalscreening

Protein‐bindingassaysProtein+moleculeinteracConbiologicalacCvity

“ReversechemicalgeneCcs”

PhenotypicacCvityassaysMolecule+organismphenotypeprotein

“ForwardchemicalgeneCcs”

Page 9: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Protein‐bindingassays

LabeledFluorescenceorradioacCvityHighthroughputTimeconsumingLabelinterference

UnlabeledLowthroughputMoreprotein/moleculetouseCosteffecCve

Page 10: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Labeledprotein‐bindingassays

Smallmoleculemicroarray

Findtargetmoleculeforaspecificprotein

Testthousandsofcompounds

Stockwell.Nature432,846‐854,2004

Page 11: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Labeledprotein‐bindingassays

Proteinmicroarray

Findwhichproteinsamoleculewillbind

Stockwell.Nature432,846‐854,2004

Page 12: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Labeledprotein‐bindingassays

DNAexpressionplasmidarrays

Chiphasplasmidsthataretakenupbyplatedcells

Testsbindingofmoleculeinalivecell(physiological)

Stockwell.Nature432,846‐854,2004

Page 13: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Labeledprotein‐bindingassays

Threehybridsystem

Bindingofcompoundtoproteinallowsexpressionofareportergene

Stockwell.Nature432,846‐854,2004

Page 14: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Unlabeledprotein‐bindingassays

Surfaceplasmonresonance

Similartosmallmoleculemicroarray,butmeasurerefracCvityofsurface

Stockwell.Nature432,846‐854,2004

Page 15: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

PhenotypicacCvity

Characterizedbasedon:

Cellularphenotype

Organismalphenotype

Page 16: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

PhenotypicacCvityassays

Lowthroughput*Mostmustbecalculatedbyhand*ThereareexcepCons

MicroarraysalsousedExaminephenotypicspecificgeneexpression

Stockwell.Nature432,846‐854,2004

Page 17: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

PhenotypeacCvityassays

ImagingAutomatedmicroscopes

RNAandproteinexpressionMaybeadvantageousoverDNAmicroarraydataforspecificproteins

Tendstobeashi,towardslessbiasedtechniques

Page 18: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Lackofspecificity—problems

Toxicity

Confoundingresults

Unexpectedresults

Dose‐dependenteffects

Page 19: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Lackofspecificity—soluCons

Useofpotentchemicals

Runlargescaleprotein‐bindingassays

ComplementresultswithRNAitechniques

Useseveralsmallmoleculeswithredundancy

StudyeachmoleculeatarangeofconcentraCons

Page 20: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Goal:UsesmallmoleculeandRNAitechniquestoidenCfyproteinsimportantincytokinesisusingDrosophila 

Page 21: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Screens

RNAi19,470dsRNAsscreenedintriplicate

Smallmolecule51,000moleculesscreened(intriplicate?)

QuanCficaConbyautomatedfluorescencemicroscopyforbinucleatecellsDouble‐checkedvisually

Page 22: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Smallmoleculescreen

25compoundsidenCfiedasmostpotentBinucleines1‐25

TestedagainatvaryingconcentraConsIniCalscreenat25μM

100μM30μM10μM

Eggert,et al.PLoS Biology 2(12),2135‐43,2004

Page 23: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Smallmoleculescreen

Alsotestedbinucleines1‐25inothercellsHeLa 16/25(64%)BSC‐1 13/25(52%)S. cerevisiae   12/25(48%)

TestedeffectsonacCnpolymerizaConInhibiConbybinucleines4,6,24,and25Binucleines24and25werecontrolsknowntodisruptmicrofilamentpolymerizaCon

Page 24: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

RNAiscreen214genesfoundEffectswereweakeroverallthanthesmallmolecules

Onenewgenefound

Eggert,et al.PLoS Biology 2(12),2135‐43,2004

Cytokinesis

Nucleicacidsynthesisanddegrada;on

Uncharacterized

Page 25: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Comparingphenotypes

RNAi Smallmolecule

Eggert,et al.PLoS Biology 2(12),2135‐43,2004

b=binucleatelc=binucleatewithlowcellcountd=binucleatewithlarge,diffuseDNAMT=binucleatewithmicrotubuleextenCons

Page 26: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Comparingphenotypes

A:binucleateB:binucleatewithlarge,diffuseDNAC:binucleatewithlowcellcountD:binucleatewithmicrotubuleextensions

RNAi

Page 27: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Comparingphenotypes

SmallmoleculeAddiConalphenotypes,includingappearanceofcleavagefurrow

Timing

GainoffuncCon

Page 28: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Pathwaystargetedbysmallmolecules

AcCncortexintegrityAssociatedwithmicrotubuleextensionphenotype

AuroraBpathwayAssociatedwithlarge,diffuseDNAphenotypeMitoCcdefect

Page 29: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

AuroraBpathway

RNAiidenCfiedAuroraB,INCENP,andSurvivininvolvementinphosphorylaCnghistoneH3andchromosomea"achmenterrorinmitosis

Foundinthechromosomalpassengercomplex

ThenewgeneidenCfiedbyRNAiscreenlocalizestothechromosomepassengercomplexCG4454,orBorealin‐related(Borr)SequencesimilaritytohumanBorealin

Page 30: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

AuroraBpathway

Arrowsindicateabsenceofphospho‐histoneH3

Page 31: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

Summary

StrongerphenotypesidenCfiedwithsmallmolecules

PhenotypeswereverysimilarbetweensmallmoleculeandRNAiscreens

CouldidenCfypathwaysdisturbedbysmallmoleculesbycomparingtoknownRNAitargetphenotypes

CouldnotdirectlyidenCfyspecifictargetsofsmallmoleculeswithmethodsused

Page 32: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

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

Page 33: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

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

Page 34: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

DrugBank

Inputsequencehere—nucleoCdeorprotein

Changematrix…PAMandBLOSUMused

Page 35: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

DrugBank

Easytofindreferences!!

Resultsshouldshowuphere,butnoluckformyproteinyet

Reminderofwhichmatrixyouchosetouse

Page 36: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

PubChem

Entryfoundbysearching“imaCnib”—searchesforbcr‐ablgivesonlyoneresult,however,andwasnotthisentry

Page 37: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

StudentquesCons

ThereviewsuggeststhatchemicalgeneCcscanhaveadvantagesovermutaConalapproachesinthatwithmutaConsyoulooseallfuncConsofaprotein,whilewithchemicalgeneCcsyoucana"ackspecificfuncCons.Isthisalwaystrue?Wouldn'titbepossibletoknock‐outonlyaspecificfuncConthroughselecCvedeleCons,orsinglebasepairmutaCons?

Notalwaystrue:canintroducemissensemutaConsinanacCvesiteforexample

Page 38: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

StudentquesCons

IntheEggertpaper,theytalkedabouthowtheconcentraConandCmingofthetreatment(whetherRNAiorsmallmolecule)canaffecttheoutcome,specificallywithrespecttotheAuroraBpathway.HasthisinformaConhelpedinunderstandingtheclinicaltrialresultsfromcancercellstreatedwithAurorainhibitorsastheypredicted?

Thismaybeagoodtheory,butgenerallyifatreatmenthasharmfuleffectsinaclinicaltrial,itiso,enabandonedaltogether.

Page 39: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

StudentquesCons

TheyalsomenConthattheythinkanaturalproductextractfromCowaniamexicanacausedagain‐of‐funcConeffectbecausenodsRNAcausedthesamephenotype.Doyouthinkthisisareasonableconclusionbasedonthis,inlightofthevariabilityinRNAiresultswehaveseenandthefactthatitisthoughtthatsuppressionofagenebyRNAimayresultinanincreaseinexpressionofothergenes?

Page 40: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

StudentquesCons

Seeingashowthecellisacomplexanddynamicenvironment,itwouldseemtometobeimpossibletoknowifthemoleculethatyouthinkyouareusing(fromyourchemicalgeneCclibrary),isactuallywhatyouareusing.Couldn'titbemodifiedbythecellanditscomponentswithoutusknowingit?

Yes!!SomedrugswetakemustbemetabolizedinourbodiesbeforetheyareactuallyacCve.

Page 41: hp://“To understand a system, you need to perturb it.” Molecular genecs techniques Mutaons Knock‐outs Knock‐ins RNAi

StudentquesCons

CanchemicalgeneCctechniquesbeespeciallyusefultoepigeneCcstudiessincemajorepigeneCcregulatorymethodsincludemethylaCon,acteylaCon,andotherchemicaladdiConstoDNA?

OneexampleofstudyofchemicalgeneCcsinepigeneCcs:genisteinfoundinsoyproductsisassociatedwithlowincidenceofcancers


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