05-筛选平台-85-中国药科大学药物筛选课件

05-筛选平台-85-中国药科大学药物筛选课件第一页，共85页。药物筛选在新药发现中的地位药物筛选的常见重要方法重要疾病治疗药物筛选方法举例展望

虚拟筛选技术（计算机科学）生物物理化学技术、组合化学分子细胞生物学天然产物化学（中草药资源）（后基因组时代药物筛选新模式）2023/5/22第二页，共85页。BiologicalTargetSelectionAssayReagentsAssayDevelopmentAndOptimizationAssayTransferAssessmentScreenDevelopmentAndValidationScreenAutomationAndOptimizationScreeningCompoundDeckLeadGenerationScreenParadigmHTS,uHTSScreenPlatforms2023/5/23第三页，共85页。III、筛选平台Beingfamiliarwithvariousscreenplatformswillbeinanadvantageouspositiontoevaluatethebestassayforthetargetscreeningwiththeidentificationofatarget.(靶标）Understandingvariousscreenplatformswillhelpinarrivingatthebestscreenswiththeavailableequipmentandreagents.（设备和试剂）2023/5/24第四页，共85页。TargetsReagentsandplatereadersavailability

AssayDesign2023/5/25第五页，共85页。1、Assayformats20世纪70年代：低通量和单一试管筛选方法；现在:(1)多孔板筛选（96－、384－、1536－，3456－，9600孔板筛选技术相继出现）(2)平板读数（platereaders)技术：荧光、发光、闪烁等检测技术的发展。

Stackersholdingseveralplates(10-40plates).2023/5/26第六页，共85页。Biacore3000，96生物分子相互作用分析系统

BiacoreS51，384962023/5/27第七页，共85页。2.Assaytechniques依据：激活或抑制、激动剂(activator)、抑制剂(inhibitor)方法:（1）体外筛选（invitro)（酶活、受体结合）（cell-free）（2）细胞水平（cell-based)（Heterogeneous&Homogeneoustypes)2023/5/28第八页，共85页。3、体外（invitro）筛选（cellfree）采用系统简单或复杂（酶反应、蛋白－蛋白相互作用、膜受体－配体结合、可溶性受体－配体结合检测实验）特点

a、被筛化合物易于直接作用于靶标；b、目标化合物作用靶标明确；c、明确的作用机理；d、易于发展便宜易得的靶标模型；e、适应新技术的发展，易于自动化。2023/5/29第九页，共85页。Invitrocell-freebiochemicalassaysHomogeneousHeterogeneousRadioactiveassaysSPAbeadSPAplateCell-freeBiochemicalAssaysChromogenicassaysNon-radioactiveassaysAbsorbanceassaysFluorescenceassaysBeadbasedassaysRadioactiveassaysNon-radioactiveassaysFiltrationassaysAdsorptionassaysPrecipitation(沉淀)assaysRadioimmunoassaysELISAassays2023/5/210第十页，共85页。A.HeterogeneousAssays多步筛选方法multipleadditions/incubations/washings/transfers/filtrations/readingsofthesignal特点Laborintensive,complicatedstep,hardtoautomate2023/5/211第十一页，共85页。（1）NonradioactiveHeterogeneousAssaysEnzymeimmunoassays(widelyusedinvitroassays)ELISA（EnzymeLinkedImmunosorbentAssay）酶联免疫吸附试验AnELISAplateAnHIVELISA,sometimescalledanHIVenzymeimmunoassay

(EIA)isthefirstandmostbasictesttodetermineifanindividualispositiveforaselectedpathogen,suchasHIV.Thetestisperformedina8cmx12cmplasticplatewhichcontainsan8x12matrix

of96wells,eachofwhichisabout1cmhighand0.7cmindiameter.

2023/5/212第十二页，共85页。PositiveELISATestNegativeELISATest

HIVantigenspre-coatedontoanELISAplatePatientserumcontainingantibodies.IfthepatientisHIV+,thenthisserumwillcontainantibodiestoHIV,andthoseantibodieswillbindtotheHIVantigensontheplate.

Anti-humanimmunoglobulincoupledtoanenzyme.Thisisthesecondantibody,anditbindstohumanantibodies.

Chromogenorsubstratewhichchangescolorwhencleavedbytheenzymeattachedtothesecondantibody.2023/5/213第十三页，共85页。SEPAntibodyAntibody-2colour,fluorescence,chemiluminescenceInhibitorScreeningSSubstrateEEnzymePProductELISA方法药物筛选原理Read!EGFR抑制剂筛选ELISA试剂盒2023/5/214第十四页，共85页。（2）RadioactiveHeterogeneousAssaysVerycommonlyused,highlysensitiveandrobustdespitehandlinghazardsandradioactivewastegeneration分离放射性产物的一般方法RadioactiveProductRadioactiveSubstractGlass-fiberfilters(filtration)WashingDryingatrt.TransferredintoavialaddscintillantCountedinascintillationcountera.FiltrationAssays2023/5/215第十五页，共85页。b.AdsorptionAssaysInproteinkinasereactionsthephosphorylatedproduct(acidic)byionicinteractioniscapturedonphosphocell-ulose（磷酸纤维素）filters;filterwashed,air-driedandtransferredintoavial;scintillantisadded,andthevialiscountedinascintillationcounter.c.PrecipitationAssaysInthetraditionalenzymeassays,theradiolabelfromthesubstrateistransferredtoaproteinacceptor,andtheradiolabeledproductisisolatedbyprecipitationwithtrichloroaceticacid(TCA,三氯乙酸);theprecipitateiscollectedbyfiltrationandwashing,thefilteristransferredintoaviral,andtheviraliscountedaftertheadditionofscintillant.2023/5/216第十六页，共85页。d.Radioimmunoassays(RIA)Aclassicalmethodformeasuring:hormones,ligandsandotherbiomolecules.抗原对其抗体进行免疫结合进行的分析通常的抗原是指人体内存在的激素，酶，小分子和多肽，特异蛋白等。对于其它动物，即异体物质，一旦把这些物质引入动物体内，其免疫系统就会作出反应，产生出专一结合抗原的抗体（即免疫球蛋白)。抗原和抗体的反应是一一对应的，高度精密专一的。

2023/5/217第十七页，共85页。B.HomogeneousAssaysOne-potassayswithnotransferorwashstepsAllthereagentsareaddedinonesteporinmultistepsThesignalisreadinaplatereader（1）RadioactiveHomogeneousAssaysBasedonscintillationproximityassay(SPA)(亲近闪烁检测)witheitherSPRbeadsorscintillant-coatedplates.Scintillationproximityassay(SPA),aninnovativeapproachforhigh-throughputscreeningintroducedin1991,allowstherapidandsensitiveassayofawidevarietyofmolecularinteractionsinahomogeneoussystem.SPAisquickandversatileand,asaresult,isnowbeingusedinthehigh-throughputscreening(HTS)laboratoriesofover60companiesworldwideastherecognizedindustrygoldstandard.2023/5/218第十八页，共85页。2023/5/219第十九页，共85页。2023/5/220第二十页，共85页。2023/5/221第二十一页，共85页。5-羟色胺肾上腺素2023/5/222第二十二页，共85页。2023/5/223第二十三页，共85页。生长激素抑制素表皮生长因子血管紧张素2023/5/224第二十四页，共85页。（2）NonradioactiveHomogeneousAssaysA.ChromogenicAssayschromophoreSubstrate(colorless)Substrate(color)max

Enzymee.g.Inthe-glucuronidase(葡糖苷酸酶)assay,thesubstratep-nitro-phenyl-glucuronide（葡糖苷酸）iscolorless,butthep-nitrophenolformedinthereactionunderalkalineconditionsiscolor-ed,andabsorbanceat415nmismeasured.2023/5/225第二十五页，共85页。B.AbsorbanceAssaysInsomereactions,thoughneitherthereactionsubstratenortheproducthasUVabsorbance,thesubstrateorproductcouldbecouldbecoupledtoanotherenzymeassaythatcanbemonitoredbyabsorbance.

EnzymeassaySubstrateproductabsorbslightintheUV/NoNo/absorbslightintheUV2023/5/226第二十六页，共85页。C.FluorescenceAssays100to1000timesmoresensitivethancolorimericorspectrophotometricassays(比色测定或

分光光度分析).PopularnonradioactivemethodsforHTSwiththeavailabilityof96-,384-wellplatereaders.2023/5/227第二十七页，共85页。2023/5/228第二十八页，共85页。2023/5/229第二十九页，共85页。2023/5/230第三十页，共85页。FluorescenceintensityassaysFluorogenicassays(CBP+PPAR)Fluorescencequenchassays(CypA+L)2023/5/231第三十一页，共85页。Fluorogenicassays

(CBP+PPAR)2023/5/232第三十二页，共85页。2023/5/233第三十三页，共85页。2023/5/234第三十四页，共85页。CPA顺十八碳四烯酸max

(410nm)PPARLigandCPAPPARmax

(410nm)2023/5/235第三十五页，共85页。2023/5/236第三十六页，共85页。2023/5/237第三十七页，共85页。2023/5/238第三十八页，共85页。Fluorescencequenchassays

(CypA+Ligand)2023/5/239第三十九页，共85页。随着DDDC838浓度增大，CypA荧光值下降，实验中，CypA浓度保持为4M，其中化合物DDDC838浓度：a,0M；b,1M;c,2M;d,4M;e,8M;f,16M;g,32M。）

2023/5/240第四十页，共85页。(ii)Fluorescencepolarization(FP)FPmeasurementsprovideinformationonmolecularorientationandmobilityandprocessesthatmodulatethem,includingreceptor–ligandinteractions,proteolysis,protein–DNAinteractions,membranefluidityandmusclecontraction.2023/5/241第四十一页，共85页。2023/5/242第四十二页，共85页。TheFPassayscanbeclassifiedintothefollowingthreedifferentmodes:1a.IncreaseinsizeMacromoleculeMacromoleculeFPSignalincreaseMacromoleculeMacromoleculeFPSignaldecrease1b.Competition2023/5/243第四十三页，共85页。2.DecreaseinsizeFPsignaldecrease3a.DirectImmunoassayPPFPsignalincrease3b.CompetitionImmunoassayPPFPsignaldecrease2023/5/244第四十四页，共85页。(ii)Fluorescenceresonanceenergytransfer(FRET)

(荧光共振能量传递)

assays(iii)Homogeneoustime-resolvedfluorescence(HTRF)/Time-resolvedFRET(iv)Fluorescencecorrelationspectroscopy(FCS)（荧光相干光谱学）(v)Fluorescencelifetimespectroscopy(FLS)2023/5/245第四十五页，共85页。2023/5/246第四十六页，共85页。2023/5/247第四十七页，共85页。2023/5/248第四十八页，共85页。2023/5/249第四十九页，共85页。2023/5/250第五十页，共85页。2023/5/251第五十一页，共85页。2023/5/252第五十二页，共85页。2023/5/253第五十三页，共85页。4、Cell-basedassaysMimictheenvironmentofalivingcell;Usedforconfirmationofleadscomingprimaryinvitrobiochemicalscreens;Usedfortargetswherebiochemicalassaysarenotavailable;Giveinformationaboutcellularinteractionswiththetargetandshedlightonthestabilityofcompounds.Traditionally,lowormediumthroughputduetothecumbersomestepsinvolved.Nowadays,HTSforprimaryscreening.HeterogeneousandHomogeneousassays2023/5/254第五十四页，共85页。CellbasedassayHomogeneousassaysHeterogeneousassaysMicrobe-basedassaysMammalianCell-basedassaysRescuetypeassaysGrowth/noGrowthassaysTwo-hybridassaysReporterassaysRadioactiveassaysNonradioactiveFunctionalassaysReporterassaysMiscellaneousassaysRadioactiveassaysFiltrationassaysRadioimmuno-assaysCytotoxicity/CellproliferationassaysNonradioactiveassaysELISAassaysCellbasedassay2023/5/255第五十五页，共85页。A.HeterogeneousAssays1.ELISAAssaysNonradioactiveassaysaremainlyELISAassys.Cellsaretreatedwithcompounds,andthecellularchangesareassayedbyELISAassays:cAMP2.RadioactiveAssaysTheassaysinvolvingradioisotopesaregenerallylimitedtoreceptor-bindingassaysandquantitationofbiomoleculeslikehormonesincellextractsbyradioimmunoassays.Filtration,Radioimmunoassays,CellProliferation2023/5/256第五十六页，共85页。B.HomogeneousAssaysThehomogeneouscell-basedassayscanbedoneinmicrobes,yeast,ormammaliancells.Theseassaysconsistofgrowingthecells,treatmentofthecellswithcompound,anddevelopingandreadingthesignal.Thehomogeneouscell-basedassayreferstotheassayinasinglestepormultiplestepadditionsinthesamewellofamicrotiterplate.2023/5/257第五十七页，共85页。1、Microbe-BasedAssaysFindantibacterialagentsandcytotoxicanticanceragents.Generally,proteinsareheterologouslyexpressedinmicrobialsystemstoobtainlargequantitiesofproteinsforbiochemicalandstructuralstudiesorforproducinglargeamountsofproteinsforclinicaluse.InclusionbodyRegainactivityaftertheyareisolated,dissolved,andrefolded.Posttranslationalmodification:glycosylation(forproteinstabilityandfortransport)Advantagesfordevelopingandrunningscreening:proteinscanbeclonedandexpressedeasilyinmicrobialcells.Cheap&simple2023/5/258第五十八页，共85页。Homologsofmanymammalianproteinsarefoundinmicrobialsystems.Manyothermammalianproteinsthatdonothavesequencehomologsbutdohavefunctionalhomologscanbeusedtocomplementfunctioninmicrobialcells.ThefunctionsofabouthalfoftheyeastandE.coligenesareknownonthebasisofaminoacidsequencesimilaritywithotherproteinsofknownfunction.Theknowledgeofthefunctionofmicrobialproteinswillhelpinelucidatingthefunctionofmanymammalianproteins:“WhatistrueforEscherichiacoliistruefortheeleohant,exceptmoreso”-----JacquesMonod.Biologicalrelevanceofmicrobialscreeningsystems2023/5/259第五十九页，共85页。a.AntibacterialactivitycompoundsInhibitionzoneTheseassayshavebeenautomatedwithrobotsundersterileenvironmentinmajorPharma.2023/5/260第六十页，共85页。b.Growth/NogrowthassaysWithfunctionalexpressionofhomologousorheterologoustargetsinmicrobialsystems,renderingthecelldependentonthetargetexpressed,agrowthornogrowth(ofthemicrobe)typeofscreencanbedeveloped.Example:S.cerevisiae—basedscreenforimmunosuppressantsImmunosuppressants,suchascyclosporinandFK506,inhibitTcellactivationandhavemadetissueandorgantransplantationareality.CyclosporinandFK506bindproteinswithpeptidylprolylisomeraseactivityandformsacomplexthatinhibitsthecalcium-calmodulinphosphatase,calcineurin.InTcellsandinyeast,freecalcineurinisessentialfortheactivationoftranscriptionfactors.Inthepresenceoftheseimmunosuppressants,yeastdosenotgrowanddivide.2023/5/261第六十一页，共85页。c.Reporter-basedassaysAtargetproteiniscoupledtoapromoter(transcriptionalfactor)thatinturniscoupledtoareporterproteinlike-galactosidase(半乳糖苷酶),luciferase(荧光素酶),orchloramphenicol(氯霉素)

acetyltransferase.ThusatargetproteinisengineeredintheextracellulardomainoftheToxRproteininE.coli.Whencompou-ndsbindtothetargetprotein,promotedimerization(二聚)ofextracellulardomainofhybridToxRprotein,whichactivatesthetoxRpromoterandconsequentlyactivatestheexpressionofreporterandcanbeeasilyreadinaplatereader.2023/5/262第六十二页，共85页。d.YeastExpressionAsyeastoffersnullbackgroundforhumanreceptors,humanGPCRsalongwithappropriatemammalianG-proteinscanbeexpressedinyeastcoupledtothepheromonesignalingpathway(信息素传导途径)toscreenforagonistsandantagonists.GPCRs

(G-proteincoupledreceptors):serotoninreceptors,dopaminereceptors,adrenergicreceptors.ThematingfactorreceptorinS.cerevisiaeiscalledSte2andissimilarinstructuretomammalianGPCRs.MammalianGPCRcanbeusedtoreplaceSte2,sothatGPCRcansignalthroughthematingfactorsignalingpathwaywhenactivatedbytheGPCRagonist.2023/5/263第六十三页，共85页。e.Two-hybridYeastsystemProtein-proteinexpression2023/5/264第六十四页，共85页。2、MammalianCell-BasedAssaysCell-basedassaysdifferfrommoretraditionalscreeningenzyme-orantibody-basedassaysinthattheuseoflivecellsrequiresspecialconsiderations.

a/freeofmycoplasma（支原体）;b/cellsfromfrozenstockshouldbeviablewithoutalterationinthegrowthcurve;c/targetproteinshouldbeexpressedatahighenoughlevelinthecells;d/littlefluctuationinreplicates…….

Cell-basedassayswilltakeseveraldaysbeforeinitiationoftheassays.Thereadoutsofhomogeneousformatareradioactive,luminescence,orfluorescence.2023/5/265第六十五页，共85页。1).RadioactiveAssaysCell-basedradioactivehomogeneousassayshavebeenusedforfunctionalassaysandreceptor-ligandassays.ReceptorbindingassaysReceptorbindingassayswithmembranereceptorscanalsobeassayswithwholecells,eitheradherentorsuspensioncells,witharadioligand.2023/5/266第六十六页，共85页。GTP--SbindingassaysG-proteinactivationcanbeassessedbymeasuringtheagonistinducedbindingofanonhydrolyzableGTPanalog[35S]GTPγStocells:SPAFlashPlates2023/5/267第六十七页，共85页。SignaltransductionassayscAMP:SPATheSPAassayisbasedoncompetitionbetweenthecellularcAMPandexogeneouslyaddedtracerof[125I]-cAMP.TheradiolabeledcAMPbindstocAMP-specificantibody,whichbindstotheSPAbeadcoatedwithsecondaryantibody.Thesignalisdetectedduetothecloseproximityoftheradiolabeltothescintillantonthebead.2023/5/268第六十八页，共85页。2).NonradioactiveAssaysCyclicAMPassaysAhighefficiencyfluorescencepolarization(HEFP)cAMPassayisahomogenouscAMPassaythatcanmeasurecAMPlevelsinwholecellsandisbasedoncompetitionbetweencAMPproducedinthecellandexogeneouslyaddedfluorescentcAMPastrancer(LJLBioSystems)2023/5/269第六十九页，共85页。Cell

Incubated

CellLysedDrugFluorescenttrancercAMP-specificantibodyFPsignalmeasurement2023/5/270第七十页，共85页。Ca2+assaysFluorescentdye:Fura-2,Indo-1Fluo-3,Fluo-4,Rhod-22023/5/271第七十一页，共85页。3).Reporter-basedAssaysMostofthetranscriptionfactorsaremodular,consistingofaDNA-bindingdomainandactivationdomain.Thesedomainscanbeinterchangedbetweendifferentfactorsandstillretaintheirfunctionalproperties.Areportergeneconstructconsistsofaninducibletrans-criptionalcontrolelementdrivingtheexpressionofareportergene.Reportergenescodeforproteinsthatpossessuniqueenzymeactivities,andtheactivityassaysareadaptabletoHTS.2023/5/272第七十二页，共85页。视黄酸受体(RAR)和视黄素X受体(RXR)由3种不同的基因：α,β和γ编码，形成了RARα，RARβ、RARγ和RXRα、RXRβ、RXRγ等多种类型的受体。受体的结构由5个亚区组成，其中高度保守的DNA结合区(DBD)和低度保守的配体结合区(LBD)发挥重要的作用。在DBD结构中，含有2个半胱氨酸丰富的锌指，它与受体识别特异的DNA序列、受体形成二聚体有关。在LBD结构中，含有大量的疏水氨基酸，它与配体的连接、转录的抑制与激活有关。可见，受体上不同的亚区行使不同的功能，由此构成受体的复杂性和功能的多样性。RAR和RXR属于类固醇和甲状腺激素受体超家族成员，它们作为配体激活的转录因子，结合到靶基因的特定应答序列(DNA)上，调节基因的转录表达。这个特定的DNA序列称为激素应答元件，视黄酸应答元件(RARE)是其中的一个典型代表，受体与RARE的结合则是维生素A信号传导的关键。研究表明，RAR本身不能形成同源二聚体而结合到RARE上，它必须要有辅助性蛋白的帮助，才能进行有效的DNA结合。1992年发现RXR就是这种辅助性蛋白，RXR通过与RAR形成异源二聚体(RAR/RXR)，增加RAR与RARE的亲和力，由此加强RAR的转录活性和对配体的敏感性。此外，RXR在其配体如9-cisRA的存在下，还可以形成同源二聚体(RXR/RXR)而结合到DNA序列上，由此介导维生素A不同的应答途径(如图)。2023/5/273第七十三页，共85页。RARRXRLBDDBDRARETTNPBLG268AGGTCAAGGTCAAGGTCADR1DR15`3`DR1LacZReportergeneSRC-1：LXXLLClampExperimentalmodelofRAR-RXRheterodimers:RARE:biotinylatedretinoic-acid-responseelement2023/5/274第七十四页，共85页。2023/5/275第七十五页，共85页。3、MiscellaneousAssaysLeadcompoundsandcompoundsofinterestforleadoptimizationareroutinelytestedforcytotoxicity,inhibitionofcytochromeP450isoenzymes(CYPs),compoundpermeabilityinCaco-2cells,andspecificityinotherrelatedassays.Profilingofthecompoundsatthetimeoftheleadoptimizationisveryhelpfulforselectingcompoundstoinvivostudies.2023/5/276第七十六页，共85页。1)CellproliferationandcytotoxicityassaysTheeffectofcompoundsonthecellisgenerallyassessedwithnonspecificcytotoxicityassays.

CellviabilitytestCellMTT(tetrazolium)LivingcellsreduceMTTtoahighlycoloredformazensaltandcanbereadinaplatereaderasanendpointreading.MTT-relativelyinsoluble(MTS/XTT)2023/5/277第七十七页，共85页。AlamarBlueAssays------isawidelyusedhomogeneouscellproliferation/cytotoxicassaythatcanbeperformedin96-or384-wellplatesinHTSmodeWhenAlamarBlueisaddedtoeitheradherentorsuspensioncellculture,thedyebecomesreducedtoahighlyfluorescentredformthatcanbereadinaspectrophotometricorfluorescencereader.Theintensityofcolororfluorescenceisproportionaltothecellviability.AlamarBlueisnontoxictocellsandallowscontinuousmonitoringofcellsatvariousperiodsoftime.2023/5/278第七十八页，共85页。2)CYPsLeadoptimizationstudiesADME/PKPromoteEarlyLeadsToCompoundsAbsorptionDistributionMetabolismExcretionPharmacokinetics2023/5/279第七十九页，共85页。3)ChipTechnologiesMicrofabricationandmicrofluidies-basedchiptechnologyisemergingandmayreplaceHTSwithfurtherminiturizationMicrochiptechnologyhasbecomeapowerfultechnologyandiswidelyusedinDNAanalysis.2023/5/280第八十页，共85页。DNAchiptechnologyWithinthedepartmentofMolecularGenetics,DNAchiptechnologyisacoreactivity.Thistechnology,whichisalsosynonymouswithDNAmicro-arrayanalysis,aimsattakingaglobalviewofgeneactivities,thusenablingther