高通量筛选解析课件

DrugDevelopmentandmodernbiotechnologyOverviewofdrugdevelopment.Procedureofdrugdevelopmentandbiotech.Onecase.Frequentproblems.Aquickquiz.Nat.Biotech.(2006)24:167BiochemicalSocietyTransaction(2006)34:313FromatargettoarealhitTargetidentificationandvalidation（药靶的发现与验证）Assayestablishment（筛选方法的建立）Assayvalidation（筛选方法的验证）Primaryscreening（初筛）Hitidentification（活性样品的发现）Hitconfirmation(secondaryscreening)

（活性样品的确定/复筛）

Arealhit!!Assayestablishment荧光偏振：以一束单一波长的偏振光照射溶液中的荧光物质，后者可吸收并释放出相应的偏振荧光。如果被激发的荧光物质处于静止状态，该物质仍将保持原有激发光的偏振性，如果其处于运动状态，该物质发出的偏振光将区别于原有激发光的偏振特性，也就是所谓荧光去偏振现象。

荧光偏振分析利用荧光偏振的原理，通过检测荧光素标记的小分子与其它分子相互作用前后分子量的变化，计算水平方向及垂直方向的荧光偏振值作相关分析。如果被检测分子大，激发时运动慢，测得的荧光偏振光值高。如果分子小，分子旋转或翻转速度快，发射光相对于激发光平面将去偏振化，测得的偏振光值低，从而计算出样品的偏振值（偏振值单位MP）。FluorescencePolarization(FP)Assay荧光偏振分析FluorescencePolarization(FP)Assay荧光偏振分析LowFPHighFPTracer:asmallbindingpartnerlabeledwithafluorescenttag.Whenexcited,itrotatesrapidly,therebydepolarizingtheemissionBinder:alargerunlabeledpartnerBindingoftwopartners:-themolecularvolumeoftracerincreases-overallrotationslowsdown-emissionlightishighlypolarizedBioluminescence

ResonanceEnergyTransfer(BRET)Assay

生物发光共振能转移分析FluorescenceResonanceEnergyTransfer(FRET)Assay

荧光共振能转移分析TimeResolved-FluorescenceAssay

时间分辨荧光ABTimeResolved-FluorescenceResonanceEnergyTransfer(TR-FRET)AssayPerkinElmer’sLanceUltrakinaseassayDonor:Eu（镧系元素）-labeledantiphospho-substrateantibodyAcceptor:Ulight-labeledsubstrateEu-AbbindstophosporylatedUlight-substrate →highTR-FRETEu-AbdoesnotbindtoUlight-substrate →lowTR-FRETAlphaScreenAlphaScreenHomogenousFlexibility:UseinanycelllineSensitivity:physiologicallyAutomatableUltraHTScompatibleMultipleassayoptionsAutomatedImagingSystem-ImageXpressMicroFromMolecularDevices(MDC)

HighContentScreening/Analysis(HCS/HCA)WhatisHighContentScreening?Cell-basedassayAutomatedmicroscopyin96or384-wellformatHighthroughputimageacquisition&processingVisualizationofsub-cellular&intracellulareventsMultiplecolors¶metersaremeasured/imageInformationrichProbes/indicators/dyesreveal:Sub-cellularlocalizationCellororganellemorphologyIonconcentration(e.g.,Ca2+,pH)MembranepotentialPathologymarkers(e.g.,apoptosis)MeasuringfluorophoresIntensitySpatialSpectralTemporalHowisitdone?100xoilobjectiveActin:green;Mitochondria:yellowNuclei:red.ImagefromMDCPlateReader-based

HTSvs.Image-basedHCSHCSaddressesneedsforassays:Multi-colors&multi-parametersLocation&quantityofmoleculesMorphologySeeingisbelieving.Apictureisworthathousandwords!NumericaldataPopulationaverage/wellImageNumericaldata(cellularandpopulation)AssayValidation

PlatelayoutAssayValidation

STABILITYANDPROCESSSTUDIES

稳定性和分析过程PLATEUNIFORMITYANDSIGNALVARIABILITYASSESSMENT

板均一性和信号变异性评估REPLICATE-EXPERIMENTSTUDY

实验可重复性STABILITYANDPROCESSSTUDIESReagentStabilityandStorageRequirementsReactionStabilityOverProjectedAssayTimeDMSOCompatabilityFermentationCompatability?B1.ReagentStabilityandStorageRequirementsItisimportanttodeterminethestabilityofreagentsunderstorageandassayconditions.Usethemanufacturerspecificationsifthereagentisacommercialproduct.Identifyconditionsunderwhichaliquotsofthereagentcanbestoredwithoutlossofactivity.testitsstabilityaftersimilarnumbersoffreeze-thawcycles.examinethestorage-stabilityofthemixtures.B2.ReactionStabilityOverProjectedAssayTimeConducttime-courseexperimentstodeterminetherangeofacceptabletimesforeachincubationstepintheassay.Thisinformationwillgreatlyaidinaddressinglogisticandtimingissues.Thestabilitystudieswillrequirerunningassaysunderstandardconditions,butwithoneofthereagentsheldforvarioustimesbeforeadditiontothereaction.Theresultswillbeusefulingeneratingaconvenientprotocolandunderstandthetoleranceoftheassaytopotentialdelays.Ifpossible,reagentsshouldbestoredinaliquotssuitablefordailyneeds.However,someinformationpertinenttosavingleftoverreagents(particularlyexpensiveones)forfutureassaysshouldbeobtained.NewlotsofcriticalreagentsshouldbevalidatedusingthebridgingstudiesB3.DMSOCompatabilityDMSOconcentrationsfrom0to10%aretested.Forcellbasedassays,itisrecommendedthatthefinal%DMSObekeptunder1%.Notethatthisstudyshouldbedonerelativelyearlyindevelopmentoftheassay.C.PLATEUNIFORMITYANDSIGNALVARIABILITYASSESSMENTOverviewAllassaysshouldhaveaplateuniformityassessment.Fornewassaystheplateuniformitystudyshouldberunover3days.Forassaytransferstheplateuniformitystudyneedbeonly2days.SummarySignalCalculationsandPlateAcceptanceCriteria

Theoverallrequirementforthesignals:therawsignalsaresufficientlytightandthereissufficientseparationbetweenthemaxandminsignalstoconductscreening.SignalCalculationsOutliers:therateofoutliers<2%.Computethemean(AVG),SD,andCV(ofthemean)foreachsignal(max,mid,min)oneachplate.

CV=(SD/n1/2)/AVGForeachofthemid-signalwells%Activity=(Wellmid–AVGmin)X(AVGmax–AVGmin)X100%%Inhibition=100-%ActivitySignalWindow(SW)orZfactor.Zfactorascreeningwindowcoefficientisdefined.Thiscoefficientisreflectiveofboththeassaysignaldynamicrangeandthedatavariationassociatedwiththesignalmeasurements,andthereforeissuitableforassayqualityassessment.TheZ-factorisadimensionless,simplestatisticalcharacteristicforeachHTSassay.TheZ-factorprovidesausefultoolforcomparisonandevaluationofthequalityofassays,andcanbeutilizedinassayoptimizationandvalidation.SummaryofAcceptanceCriteria(1)Intra-plateTests(eachplate):

CVmaxandCVmid≤20%

变异系数（coefficientofvariation），亦称离散系数（coefficientofdispersion） 或相对偏差(rsd)

CVmin≤20%orSDmin≤(SDmid,SDmax)NormalizedSDmid≤20SW≥2orZ≥0.4.SpatialUniformityAssessmentDrift（漂移效应）: significanttrendsinthesignalfromleft-to-rightandtop-to-bottomEdgeeffects（边缘效应）.DriftandEdgeEffectsUniform-SignalformatNomaterialedge,driftorotherspatialeffects.

Lessthan20%ofAVGInter-plateandInter-DayTests:Thenormalizedaveragemid-signalshouldnottranslateintoafoldshift

>2withindays,

>2acrossanytwodays.SummaryofAcceptanceCriteria(2)PrimaryScreeningMcGill’sCompoundLibrariesCompoundStorage123,900Total1,120Prestwick~1,000Chemistcollaborators40,000ChembridgeDiscoverset10,000ChembridgeDIVERSet1,280SigmaLOPAC500BIOMOL50,000Maybridge16,000MaybridgeHitFinder2,000MSD2,000MSDspectrum#CompoundsSourceLiquidHandling

Realroboticsystems:Humanrobot:expensivebuthighthroughputcheapbutlowthroughputPlateReadersFluorescenceintensityFluorescencepolarizationTime-resolvedfluorescenceLuminescenceAbsorbanceScintillation

AssaysofvariousreadoutsDrugdiscoverytoday11:718Hitidentification

DataMining Theexplorationandanalysisoflargequantitiesofdatatodiscovermeaningfulpatternsandrules.DataMiningAssayanddataqualityPrioritizationofpossibleactives Physicalproperties---drug-likeness ADME-Toxproperties---PK,toxicity.. Scaffolds/structuralfingerprints.RapidEliminationofSwill(REOS)/Rule-of-5HitconfirmationConfirmatoryscreen:secondaryscreen（复筛）

Confirmedhitsaretestedinafunctionalassay.Membranepermeabilityisusuallyacriticalparameter.

Counterscreen（对抗筛选？）

usuallyincludedrugtargetsofthesameproteinorreceptorfamily,decreasestheriskofso-calledoff-targetsideeffects,themechanismofaction.

Orthogonaltesting:（正交测试）

Confirmedhitsareassayedusingadifferentassaywhichisusuallyclosertothetargetphysiologicalconditionorusingadifferenttechnology.HitconfirmationDoseresponse(IC50)（量效关系）Cytotoxicity（毒性）Selectivity（选择性）Mechanisticstudies（机制）HitconfirmationNuisance（有害）HitsSteepdose-responsecurvesFlatstructure-activityrelationshipsHighsensitivitytoassayconditions……AggregatinginhibitorsAggregatinginhibitorsFromatargettoarealhitTargetidentificationandvalidationAssayestablishmentAssayvalidationPrimaryscreeningHitidentificationHitconfirmation(secondaryscreening) Arealhit!!Case:ScreeningcompoundsinhibitingVif-mediatedhA3GdegradationAmemberofanRNA/DNAcytidinedeaminaseAPOBECsuperfamily(apolipoproteinBmRNA-editingenzymecatalyticpolypeptide).HumanAPOBEC3G(hA3G)hA3G164101256298384--HxE-----CxxxC---HxE-----CxxxC-CD1CD2Zinc++coordinationmotifInhibitsHIV-1replicationintheabsenceofVif.(2002)Nature.418(6898):646-50Zhangetal.(2003)Nature.424(6944):94Mangeatetal.(2003)Nature.424(6944):99Lecossier(2003)Science.300(5622):1112Harrisetal.(2003)Cell.113(6):803RebeccaK,etal.TrendsBiochemSci.2007;32(3):118-28.HumanAPOBEC3GandHIV-1TheEndAPOBEC3G-Vif-Cul5-SCF(Skp1-cullin-F-box)complexAPOBEC3G-VifModelAPOBEC3GYFPVifAPOBEC3GYFP492nm520nmVifAPOBEC3GYFPVifInhibitor26SProteasomepolyubiquitination

pathwaydegradationNosignalNormal＋＋SignalingInhibitionofVif-mediatedhA3GdegradationbyIMB-26/35IMB-26/35donotinhibitE4orf6-mediatedp53degradationp53YFPp53YFP492nm520nmp53YFPInhibitor26SProteasomepolyubiquitinationdegradationNosignalNormalE4orf6＋SignalingE4orf6AntiviralmechanismofIMB-26/35AntiviraleffectofIMB-26/35uponHIV-1producedfrom293TwithorwithoutAPOBEC3G293TCo-transfectHIV-1InfectHIV-1hA3GorpcDNA+TZM-blWith/withouttreatmentInhibitionofHIV-1replicationinT-celllineshA3GmRNA;1.000.240.02IMB-26/35didnotinhibitHIV-1RT,PR,andIN.PRactivityassayRTactivityassayINactivityassayAntiviralmechanismofIMB-26/35Vif(SLQ)hA3GGFPRLUC395nm395nm510nmBRET-basedinvivoAssayXLuciferin510nm395nmWavelengthFluorescenceDeepBlueCTheprimarytargetofIMB-26/35BIAcoreassayisusedtodeterminethebindingaffinityofIMB-26/35toeitherViforhA3G.HIV-1VIFhA3GHIV-1VIFHIV-1HIV-1HIV-1HIV-1VIFhA3GIMB-26/35HIV-1hA3GIMB-26/35VIFhA3GIMB-26/35AntiviralMechanismofIMB-26/35Frequentproblems:TargetCompoundsAssayqualityPrimaryscreen+function≠arealhit!Data