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上讲小结人类基因组计划药物靶点Lead药物HitHTS，化学合成（盲筛、效率低下)分子生物学结构生物学靶标结构计算机辅助药物设计化学合成/HTS效率高药物设计高通量筛选技术关键第二章、药物设计和筛选中国科学院上海药物研究所“药物发现与设计中心”http://www.

dddc.ac.cn沈旭•药物筛选在新药发现中的地位•药物筛选的常见重要方法（结合本实验室工作）•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）新药筛选基本方法一、随机筛

选•最经典筛选方式。曾发挥重要作用,50～60年代较为变通。•它用一个或多种生物试验手段评筛化合物或自然资源。现在应用的许多有效治疗药物都是通过随机筛选得到的随机筛选典型代表•细菌培养法从自然资源中筛选抗菌素;•瘤株细胞培养法从多种来源的化学物质

中筛选抗癌药物；７０年代后利用受体（竞争）结合法随机筛选神经－精神性药物等。随机筛选遭到冷落，新药研制部门特别是药物产业部门很少依靠随机筛选做为发现新药的主要手段二、经验式重复筛选使用最为广泛的传统的筛选方法主要特点：•合成筛选紧密配合，经重

复性（trialanderror）试验过程，根据构效关系（ＳＡＲ），不断对化合物进行结构修饰（structuralmodification）,找到选择性导向或候选化合物。•属于定向合成与筛选，成败的关

键在于确定适当筛选目标和范围，选好靶标（target）和筛选指标。除去抗感染、抗病毒和抗寄生虫类药物以外，制药工业用来做为药物靶向筛选的靶标已有４１７种，包括受体、酶、离子通道等，涉及１０个系统的功能（表）。在人类基因组计划完成后这种靶标可增至３０００－４０００

个。据统计，人类疾病有３０,０００种，但药物能控制和改善的只有１００－１５０种。未得到控制和改善的疾病大多数与基因有关，每一种疾病大约涉及５－１０个基因。因此，靶向筛选是新药筛选的一个很有发展潜力的重要方向和领域。(

后基因组时代药物发展新模式）三、药物合理设计与筛选•基于结构的药物设计与筛选(structure-baseddrugdesignandscreening)•将基于机制的筛选与药物合理设计融合为一体，为了进行药物合理设计，必须获得靶标大分子的结构，特别是

三维结构的信息。因此，该种筛选策略的第一步是靶标的分子结构分析。在实施中也可取已知三维结构的靶标，直接进行药物合理设计，做为工作的起始点。如果大分子一级结构已知，三维结构尚水阐明者，可根据同源蛋白模建等方法预测其三维结构。大分子

一级结构不清，只有配体结构信息的，根据其配体推导药效基因(pharmacophore)模型，或提出假想受体，间接设计化合物。•理论计算－药物设计－化学合成－生物筛选密切配合成功的药物合理设计可大量减少化学合成和生物筛选

的工作量，提高新药发现的机率，降低基金投入。“计算机虚拟筛选”与“高通量筛选”技术完美结合大大加速了新药发现进程临床前研究临床研究新药Summary：药物筛选▪分子生物学、结构生物学、基因组、蛋白质组、组合化学、高通量筛选、计算

机辅助药物设计、生物信息学与化学信息学(数据库学)、化学生物学化合物药物发现II、药物筛选HitsScreenLeadsPre-clinicDevelopmentClinicalDevelopmentScreenParadigmBlock-busterDrugsBiologicalTarget

SelectionAssayReagentsAssayDevelopmentAndOptimizationAssayTransferAssessmentScreenDevelopmentAndValidationScreenAut

omationAndOptimizationScreeningCompoundDeckLeadGenerationScreenParadigmHTS,uHTSScreenPlatforms高通量筛选技术（High-ThroughputScreen,HTS)(tobecon

tinued)(continued)III、筛选平台•Beingfamiliarwithvariousscreenplatformswillbeinanadvantageouspositiontoevaluatethebestassayforthetargetsc

reeningwiththeidentificationofatarget.(靶标）•Understandingvariousscreenplatformswillhelpinarrivingatthebestscreenswiththeavailableequipmentandreag

ents.（设备和化合物）TargetsReagentsandplatereadersavailabilityAssayDesign1、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).Biacore3000，96BiacoreS5

1，38496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)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。Invi

trocell-freebiochemicalassaysHomogeneousHeterogeneousRadioactiveassaysSPAbeadSPAplateCell-freeBiochemicalAssaysChromogenicassaysNon-radioact

iveassaysAbsorbanceassaysFluorescenceassaysBeadbasedassaysRadioactiveassaysNon-radioactiveassaysFiltrationassaysAdsorptiona

ssaysPrecipitationassaysRadioimmunoassaysELISAassaysA.HeterogeneousAssays多步筛选方法multipleadditions/incubations/washings/transfers/f

iltrations/readingsofthesignal特点Laborintensive,complicatedstep,hardtoautomate（1）NonradioactiveHeterogeneousAssaysEnzymeimmunoassays(widelyu

sedinvitroassays)ELISA（EnzymeLinkedImmunosorbentAssay）酶联免疫吸附试验AnELISAplateAnHIVELISA,sometimescalledanHIVenzymeimmunoassay(EIA)isth

efirstandmostbasictesttodetermineifanindividualispositiveforaselectedpathogen,suchasHIV.Thetestisperformedina8cmx12cmpl

asticplatewhichcontainsan8x12matrixof96wells,eachofwhichisabout1cmhighand0.7cmindiameter.PositiveELISATestNegativeE

LISATestHIVantigenspre-coatedontoanELISAplatePatientserumcontainingantibodies.IfthepatientisHIV+,thenthisserumwillcontainantibodiest

oHIV,andthoseantibodieswillbindtotheHIVantigensontheplate.Anti-humanimmunoglobulincoupledtoanenzyme.Thisisthesecondantibod

y,anditbindstohumanantibodies.Chromogenorsubstratewhichchangescolorwhencleavedbytheenzymeattachedtothesecondantibody.SEPA

ntibodyAntibody-2colour,fluorescence,chemiluminescenceInhibitorScreeningSSubstrateEEnzymePProductELISA方法药物筛选原理Read!EGFR抑制剂筛选ELISA试剂

盒（2）RadioactiveHeterogeneousAssaysVerycommonlyused,highlysensitiveandrobustdespitehandlinghazardsandradioactivewaste

generation分离放射性产物的一般方法RadioactiveProductRadioactiveSubstractGlass-fiberfilters(filtration)WashingDryingatrt.Transferred

intoavialaddscintillantCountedinascintillationcountera.FiltrationAssaysb.AdsorptionAssaysInproteinkinasereactionsthephosphorylatedprodu

ct(acidic)byionicinteractioniscapturedonphosphocell-ulose（磷酸纤维素）filters;filterwashed,air-driedandtransferredintoavial;scintillantisadd

ed,andthevialiscountedinascintillationcounter.c.PrecipitationAssaysInthetraditionalenzymeassays,theradiolabelfromthesubstrateistransferredtoaprotein

acceptor,andtheradiolabeledproductisisolatedbyprecipitationwithtrichloroaceticacid(TCA);theprecipitateiscollectedbyfiltrationandwashing,thefilteri

stransferredintoaviral,andtheviraliscountedaftertheadditionofscintillant.d.Radioimmunoassays(RIA)Aclassical

methodformeasuring: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。B.HomogeneousAssays•One-potassayswithnotransferorwashsteps•Allthereagentsareaddedinonestepo

rinmultisteps•Thesignalisreadinaplatereader（1）RadioactiveHomogeneousAssaysBasedonscintillationproxim

ityassay(SPA)(亲近闪烁检测)witheitherSPRbeadsorscintillant-coatedplates.Scintillationproximityassay(SPA),aninnovativeapproachfo

rhigh-throughputscreeningintroducedin1991,allowstherapidandsensitiveassayofawidevarietyofmolecularinteractionsinahomogeneoussystem.SPAis

quickandversatileand,asaresult,isnowbeingusedinthehigh-throughputscreening(HTS)laboratoriesofover60companiesworldwideastherec

ognizedindustrygoldstandard.（2）NonradioactiveHomogeneousAssaysA.ChromogenicAssayschromophoreSubstrate(colorless)Substrate(color)maxEnzymee.g.Int

he-glucuronidase(葡糖苷酸酶)assay,thesubstratep-nitro-phenyl-glucuronide（葡糖苷酸）iscolorless,butthep-nitrophenolformedinthereactionunderalkalinecondit

ionsiscolor-ed,andabsorbanceat415nmismeasured.B.AbsorbanceAssaysInsomereactions,thoughneitherthereactionsubstratenortheproducthasUVabsor

bance,thesubstrateorproductcouldbecoupledtoanotherenzymeassaythatcanbemonitoredbyabsorbance.EnzymeassaySubstrateproductab

sorbslightintheUV/NoNo/absorbslightintheUVC.FluorescenceAssays•100to1000timesmoresensitivethancolorimeric

orspectrophotometricassays.•PopularnonradioactivemethodsforHTSwiththeavailabilityof96-,384-wellplatereaders.(i)Fluorescenceintensitya

ssays•Fluorogenicassays(CBP+PPAR)•Fluorescencequenchassays(CypA+L)•Fluorogenicassays(CBP+PPAR)CPAmax(410nm)PPARLigandCPAPPARmax(410nm)HOOci

s-Parinaricacid01234020406080100Fluorescence/A.U.CPPARLBD/MFluorescencequenchassays(CypA+Ligand)FAnmabCdefg随着DD

DC838浓度增大，CypA荧光值下降，实验中，CypA浓度保持为4M，其中化合物DDC838浓度：a,0M；b,1M;c,2M;d,4M;e,8M;f,16M;g,32M。）(ii)

Fluorescencepolarization(FP)FPmeasurementsprovideinformationonmolecularorientationandmobilityandprocessesthatmodulatethem,i

ncludingreceptor–ligandinteractions,proteolysis,protein–DNAinteractions,membranefluidityandmusclecontraction.TheFPassayscanbecl

assifiedintothefollowingthreedifferentmodes:1a.Increaseinsize+MacromoleculeMacromoleculeFPSignalincrease+Macromolecule++MacromoleculeFPSignald

ecrease1b.Competition2.Decreaseinsize+FPsignaldecrease3a.DirectImmunoassayPPFPsignalincrease3b.CompetitionImmunoassa

yPP+FPsignaldecrease(ii)Fluorescenceresonanceenergytransfer(FRET)(荧光共振能量传递)assays(iii)Homogeneoustime-resol

vedfluorescence(HTRF)/Time-resolvedFRET(iv)Fluorescencecorrelationspectroscopy(FCS)（荧光相干光谱学）(v)Fluorescenceli

fetimespectroscopy(FLS)4、Cell-basedassays•Mimictheenvironmentofalivingcell;•Usedforconfirmationofleadscomingprimaryin

vitrobiochemicalscreens;•Usedfortargetswherebiochemicalassaysarenotavailable;•Giveinformationaboutcellularinteraction

swiththetargetandshedlightonthestabilityofcompounds.Traditionally,lowormediumthroughputduetothecumbersomestepsinvolved.Nowadays,HTSforprimarys

creening.HeterogeneousandHomogeneousassaysCellbasedassayHomogeneousassaysHeterogeneousassaysMicrobe-basedassaysMammalianCell-b

asedassaysRescuetypeassaysGrowth/noGrowthassaysTwo-hybridassaysReporterassaysRadioactiveassaysNonradioactiveFunctionalassaysReporterassays

MiscellaneousassaysRadioactiveassaysFiltrationassaysRadioimmuno-assaysCytotoxicity/CellproliferationassaysNonradioactiveassaysELI

SAassaysCellbasedassayA.HeterogeneousAssays1.ELISAAssaysNonradioactiveassaysaremainlyELISAassys.Cellsaretreatedwithco

mpounds,andthecellularchangesareassayedbyELISAassays.2.RadioactiveAssaysTheassaysinvolvingradioisoto

pesaregenerallylimitedtoreceptor-bindingassaysandquantitationofbiomoleculeslikehormonesincellextractsbyradioimmunoassays.Filtration,Radioimmunoassays

,CellProliferationB.HomogeneousAssaysThehomogeneouscell-basedassayscanbedoneinmicrobes,yeast,ormammaliancells.Theseas

saysconsistofgrowingthecells,treatmentofthecellswithcompound,anddevelopingandreadingthesignal.Thehomogeneouscel

l-basedassayreferstotheassayinasinglestepormultiplestepadditionsinthesamewellofamicrotiterplate.1、Microbe-BasedAssays•Finda

ntibacterialagentsandcytotoxicanticanceragents.Inclusionbody,posttranslationalmodificationcheap,simplea.AntibacterialactivitycompoundsInhibition

zoneTheseassayshavebeenautomatedwithrobotsundersterileenvironmentinmajorPharma.b.Growth/NogrowthassaysWithfunctionalexpressionofhomologousor

heterologoustargetsinmicrobialsystems,renderingthecelldependentonthetargetexpressed,agrowthornogrowth(ofthemicrobe)typeofsc

reencanbedeveloped.c.Reporter-basedassaysAtargetproteiniscoupledtoapromoter(transcriptionalfactor)that

inturniscoupledtoareceptorproteinlike-galactosidase,luciferase,orchloramphenicolacetyltransferase.Thusatargetpro

teinisengineeredintheextracellulardomainoftheToxRproteininE.coli.Whencompoundsbindtothetargetprotein,promotedimerizat

ionofextracellulardomainofhybridToxRprotein,whichactivatesthetoxRpromoterandconsequentlyactivatestheexpressionofreporterandcanbeeasil

yreadinaplatereader.d.YeastExpressionAsyeastoffersnullbackgroundforhumanreceptors,humanGPCRsalongwithappropriatemam

malianG-proteinscanbeexpressedinyeastcoupledtothepheromonesignalingpathway(信息素传导途径）toscreenforagonistsandantagonists.e.Two

-hybridYeastsystemProtein-proteinexpression2、MammalianCell-BasedAssays•Cell-basedassaysdifferfrommoret

raditionalscreeningenzyme-orantibody-basedassaysinthattheuseoflivecellsrequiresspecialconsiderations.a/f

reeofmycoplasma（支原体）;b/cellsfromfrozenstockshouldbeviablewithoutalterationinthegrowthcurve;c/targetproteinsho

uldbeexpressedatahighenoughlevelinthecells;d/littlefluctuationinreplicates…….•Cell-basedassayswilltakesever

aldaysbeforeinitiationoftheassays.•Thereadoutsofhomogeneousformatareradioactive,luminescence,orfluorescence.1)

.RadioactiveAssaysCell-basedradioactivehomogeneousassayshavebeenusedforfunctionalassaysandreceptor-ligandassays.Receptorbindi

ngassaysReceptorbindingassayswithmembranereceptorscanalsobeassayswithwholecells,eitheradherentorsuspensioncells,with

aradioligand.GTP--Sbindingassays,Signaltransductionassays2).NonradioactiveAssaysCyclicAMPassaysAhigheffici

encyfluorescencepolarization(HEFP)cAMPassayisahomogenouscAMPassaythatcanmeasurecAMPlevelsinwholecellsandisbasedonco

mpetitionbetweencAMPproducedinthecellandexogeneouslyaddedfluorescentcAMPastrancer(LJLBioSystems)CellIncuba

tedCellLysedDrugFluorescenttrancercAMP-specificantibodyFPsignalmeasurement3).Reporter-basedAssays•Mostofthetranscriptionfactorsaremodular,consis

tingofaDNA-bindingdomainandactivationdomain.Thesedomainscanbeinterchangedbetweendifferentfactorsandstillretaintheirfunctionalproperties

.•Areportergeneconstructconsistsofaninducibletrans-criptionalcontrolelementdrivingtheexpressionofareportergene.•Reportergenescodefo

rproteinsthatpossessuniqueenzymeactivities,andtheactivityassaysareadaptabletoHTS.RARRXRLBDDBDRARETTNPBLG268AGGTCA

AGGTCAAGGTCADR1DR15`3`DR1LacZReportergeneSRC-1：LXXLLClampExperimentalmodelofRAR-RXRheterodimers:RARE:b

iotinylatedretinoic-acid-responseelement3、MiscellaneousAssays•Leadcompoundsandcompoundsofinterestforleadoptimi

zationareroutinelytestedforcytotoxicity,inhibitionofcytochromeP450isoenzymes(CYPs),compoundpermeabilityinCaco-2cells,andspecificityin

otherrelatedassays.•Profilingofthecompoundsatthetimeoftheleadoptimizationisveryhelpfulforselectingcompoundstoinvivostudies.1)Cell

proliferationandcytotoxicityassaysTheeffectofcompoundsonthecellisgenerallyassessedwithnonspecificcytotoxicityassays.Cellviabilitytes

tCellMTT(tetrazolium)LivingcellsreduceMTTtoahighlycoloredformazensaltandcanbereadinaplatereaderasanendpointread

ing.MTT-relativelyinsoluble(MTS/XTT)AlamarBlueassays2)CYPsLeadOptimizationstudiesADME/PKPromoteEarlyLeadsToCompounds•Absor

ption•Distribution•Metabolism•Excretion•Pharmacokinetics3)ChipTechnologies•Microfabricationandmicrofluidies-basedchiptechnologyisemergingand

mayreplaceHTSwithfurtherminiturization•MicrochiptechnologyhasbecomeapowerfultechnologyandiswidelyusedinDNAanalysis.DNAchiptechnologyWithinthedepartm

entofMolecularGenetics,DNAchiptechnologyisacoreactivity.Thistechnology,whichisalsosynonymouswithDNAmicro-arrayanalysis,aimsatt

akingaglobalviewofgeneactivities,thusenablingtheresearchertosimultaneouslyfollowtheexpressionlevelsofthousandsofgenesinasing

leexperiment.Thepurposeofthisistogainmolecularunderstandingofthebiologicalactionofdrugcandidatemoleculestohelpselectanddevelopop

timaldrugs.Furthermore,throughglobalgeneexpressionanalysisindiseasemodels/treatmentsystems,itisalsopossibletoidentifynoveltargetsforfuturedrugdevelo

pment.AveryimportantaspectofDNAchiptechnologyistheabilitytoidentifybiomarkersthatcanbeusedtoreadilymonitordrugefficaciesaswellassideeffectsanddisea

seprogression.DNAchipanalysisisarecentlydevelopedtechnologythatisstillbeingshaped.Currently,anumberofdifferentplatforms(technicalmethods)a

rebeingused.Inourset-upweprintcDNAmoleculesontomicroscopeglassslidesusingaroboticinstrument.Theslidesarehybridisedwithdualdyelabelledsamplesderiv

edfromcontrolandtreatedspecimensandtheexpressionlevelsaremeasuredasthedegreeofhybridisationbylaserdensitometrys

canning.Thesedataarethenonagenebygenebasiscomparedanditcanbedeterminedwhetherandhowmucheachindividualgeneassayedwasregulatedinthe

experiment.Theamountsofdataderivedfromjustasingleexperimentareverylarge-anissuethatiscommonlyreferredtoasthedatadelugeofthistechnology.

OneofthechallengesofDNAchiptechnologyhasbeentodeveloptoolsforhandlingandminingtheselargedatasets.Thus,statisticalandbio

-informaticdataanalysisarekeycomponentsofDNAchiptechnology.Thegroupofpeopleinvolvedintheuseofthistechnologyisthereforecomposedofmolecularbiolog

ists,biochemists,statisticiansaswellasbio-informaticians.SummaryHumangenomeMol.TargetNumberHTSAssaysAdapatation•Tar

getteam(screeningplatform)•HTSgroups(automationscreen)•Genomic(targetsnumber,compounddecksize)•Combinatorialchem

icalsynthesisBasepair3billionGenes30,000RobustHomogeneousHTSassaysSuggestedReferences1.P.Fernandes,“Technologi

caladvancesinhighthroughputscreening”,CurrentOpinionChemBiol,1998,2,597.2.M.Meza,“Bead-basedHTSapplicationsindrugdiscovery”,DrugDiscover

yToday,2000,1,38.3.J.Drews,“Drugdiscoverytoday-andtomorrow”,DrugDiscoveryToday,2000,5,2.4.R.SeethalaandP.Fernandes(Editors),“HandbookofDrugScree

ning”,MarcelDekker,Inc.2001.NY.Basel.