中国药物筛选平台（ScreeningPlatforms）

1、中国药物筛选平台（Screening Platforms）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）Biological Target SelectionAssay ReagentsAssay DevelopmentAnd OptimizationAssay TransferAssessmentScreen DevelopmentAnd ValidationScreen AutomationAnd Optimization

2、Screening Compound DeckLead GenerationScreen ParadigmHTS, uHTSScreen PlatformsIII、筛选平台Being familiar with various screen platforms willbe in an advantageous position to evaluate the bestassay for the target screening with the identificationof a target. (靶标）Understanding various screen platforms will

3、 help in arriving at the best screens with the available equipment and reagents. （设备和试剂）TargetsReagents and plate readers availability Assay Design1、 Assay formats20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（plate readers) 技术：荧光、发光、闪烁等 检测技术的发展。 Stackers holding severa

4、l plates (10-40 plates).Biacore 3000，96生物分子相互作用分析系统 Biacore S51，38496 2. Assay techniques依据：激活或抑制、 激动剂(activator)、抑制剂 (inhibitor)方法:（1）体外筛选（in vitro)（酶活、受体结合） （cell-free）（2）细胞水平（cell-based)（Heterogeneous & Homogeneous types) 3、体外（in vitro）筛选 （cell free）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

5、 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。In vitro cell-free biochemical assaysHomogeneousHeterogeneousRadioactiveassaysSPA beadSPA plateCell-free Biochemical AssaysChromogenic assaysNon-radioactiveassaysAbsorbance assaysFluorescence assaysBead based assaysRadioacti

6、veassaysNon-radioactiveassaysFiltration assaysAdsorption assaysPrecipitation (沉淀) assaysRadioimmunoassaysELISA assaysA. Heterogeneous Assays多步筛选方法 multiple additions/incubations/washings/transfers/filtrations/readings of the signal特点Labor intensive, complicated step, hard to automate（1）Nonradioactiv

7、e Heterogeneous AssaysEnzyme immunoassays (widely used in vitro assays)ELISA （Enzyme Linked Immunosorbent Assay）酶联免疫吸附试验An ELISA plateAn HIV ELISA, sometimes calledan HIV enzyme immunoassay (EIA) is the first and most basic test to determine if an individual is positive for a selected pathogen, such

8、 as HIV. The test is performed in a 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which is about 1 cm high and 0.7 cm in diameter. Positive ELISA TestNegative ELISA Test HIV antigens pre-coated onto an ELISA platePatient serum containing antibodies. If the patient i

9、s HIV+, then this serum will contain antibodies to HIV, and those antibodies will bind to the HIV antigens on the plate. Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies. Chromogen or substrate which changes color when cleaved by the enzym

10、e attached to the second antibody.SEPAntibodyAntibody-2colour, fluorescence, chemiluminescenceInhibitor ScreeningSSubstrateEEnzymePProductELISA 方法药物筛选原理Read !EGFR 抑制剂筛选ELISA试剂盒（2）Radioactive Heterogeneous AssaysVery commonly used, highly sensitive and robustdespite handling hazards and radioactive w

11、aste generation分离放射性产物的一般方法RadioactiveProductRadioactiveSubstractGlass-fiber filters(filtration)WashingDrying at rt.Transferred into a vialaddscintillantCounted in a scintillationcountera. Filtration Assaysb. Adsorption AssaysIn protein kinase reactions the phosphorylated product(acidic) by ionic in

12、teraction is captured on phosphocell-ulose （磷酸纤维素）filters; filter washed, air-dried and transferred into a vial; scintillant is added, and the vial is counted in a scintillation counter.c. Precipitation AssaysIn the traditional enzyme assays, the radiolabel from the substrate is transferred to a pro

13、tein acceptor, and the radiolabeled product is isolated by precipitation withtrichloroacetic acid (TCA, 三氯乙酸); the precipitate is collected by filtration and washing, the filter is transferred into a viral,and the viral is counted after the addition of scintillant.d. Radioimmunoassays(RIA)A classica

14、l method for measuring:hormones, ligands and other biomolecules.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. Homogeneous Assays One-pot assays with no transfer or wash steps All the reagents are added in one st

15、ep or in multisteps The signal is read in a plate reader（1）Radioactive Homogeneous AssaysBased on scintillation proximity assay (SPA) (亲近闪烁检测) with either SPR beads or scintillant-coated plates.Scintillation proximity assay (SPA), an innovative approach for high-throughput screening introduced in 19

16、91, allows the rapid and sensitive assay of a wide variety of molecular interactions in a homogeneous system. SPA is quick and versatile and, as a result, is now being used in the high-throughput screening (HTS) laboratories of over 60 companies worldwide as the recognized industry gold standard.5-羟

17、色胺肾上腺素生长激素抑制素表皮生长因子血管紧张素（2）Nonradioactive Homogeneous AssaysA. Chromogenic AssayschromophoreSubstrate (colorless)Substrate (color)max Enzymee.g. In the -glucuronidase(葡糖苷酸酶) assay, the substrate p-nitro-phenyl -glucuronide（葡糖苷酸）is colorless, but the p-nitrophenol formed in the reaction under alkalin

18、e conditions is color-ed, and absorbance at 415 nm is measured.B. Absorbance AssaysIn some reactions, though neither the reaction substrate nor the product has UV absorbance, the substrate or product could be could be coupled to another enzyme assay that can be monitored by absorbance. Enzyme assayS

19、ubstrateproductabsorbs light in the UV/NoNo /absorbs light in the UVC. Fluorescence Assays100 to 1000 times more sensitive than colorimeric or spectrophotometric assays (比色测定 或 分光光度分析). Popular nonradioactive methods for HTSwith the availability of 96-, 384-well plate readers.Fluorescence intensity

20、assaysFluorogenic assays (CBP + PPAR)Fluorescence quench assays (CypA + L)Fluorogenic assays (CBP + PPAR)2022/7/19332022/7/1934CPA顺十八碳四烯酸max (410 nm)PPARLigandCPAPPARmax (410 nm)2022/7/19372022/7/1938Fluorescence quench assays (CypA + Ligand)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,

21、0 M；b, 1M; c, 2 M; d, 4 M; e, 8 M; f, 16 M; g, 32 M。） (ii) Fluorescence polarization (FP) FP measurements provide information on molecular orientation and mobility and processes that modulate them, including receptorligand interactions, proteolysis, proteinDNA interactions, membrane fluidity and mus

22、cle contraction. The FP assays can be classified into the followingthree different modes:1a. Increase in sizeMacro moleculeMacro moleculeFPSignal increaseMacro moleculeMacro moleculeFPSignal decrease1b. Competition2. Decrease in sizeFP signal decrease3a. Direct ImmunoassayPPFP signal increase3b. Com

23、petition ImmunoassayPPFP signal decrease(ii) Fluorescence resonance energy transfer (FRET) (荧光共振能量传递) assays (iii) Homogeneous time-resolved fluorescence (HTRF)/ Time-resolved FRET (iv) Fluorescence correlation spectroscopy (FCS) （荧光相干光谱学）(v) Fluorescence lifetime spectroscopy (FLS)2022/7/19462022/7

24、/19474、Cell-based assaysMimic the environment of a living cell;Used for confirmation of leads coming primary in vitro biochemical screens;Used for targets where biochemical assays are not available;Give information about cellular interactions with the target and shed light on the stability of compou

25、nds.Traditionally, low or medium throughput due to the cumbersome steps involved.Nowadays, HTS for primary screening.Heterogeneous and Homogeneous assaysCell based assayHomogeneous assaysHeterogeneous assaysMicrobe-based assaysMammalianCell-based assaysRescue typeassaysGrowth/no Growth assaysTwo-hyb

26、ridassaysReporter assaysRadioactiveassaysNonradioactiveFunctional assaysReporter assaysMiscellaneous assaysRadioactiveassaysFiltration assaysRadioimmuno- assaysCytotoxicity/Cellproliferation assaysNonradioactiveassaysELISAassaysCell based assayA. Heterogeneous Assays1. ELISA AssaysNonradioactive ass

27、ays are mainly ELISA assys.Cells are treated with compounds, and the cellular changes are assayed by ELISA assays: cAMP 2. Radioactive AssaysThe assays involving radioisotopes are generally limited to receptor-binding assays and quantitation of biomolecules like hormones in cell extracts by radioimm

28、unoassays.Filtration, Radioimmunoassays, Cell ProliferationB. Homogeneous AssaysThe homogeneous cell-based assays can be done in microbes, yeast, or mammalian cells. These assaysconsist of growing the cells, treatment of the cells with compound, and developing and reading thesignal. The homogeneous

29、cell-based assay refers tothe assay in a single step or multiple step additionsin the same well of a microtiter plate.2022/7/19581、Microbe-Based AssaysFind antibacterial agents and cytotoxic anticancer agents.Generally, proteins are heterologously expressed in microbial systems to obtain large quant

30、ities of proteins for biochemical and structural studies or for producing large amounts of proteins for clinical use.Inclusion bodyRegain activity after they are isolated, dissolved, and refolded.Posttranslational modification: glycosylation (for protein stability and for transport)Advantages for de

31、veloping and running screening: proteins can be cloned and expressed easily in microbial cells.Cheap & simple2022/7/1959Homologs of many mammalian proteins are found in microbial systems.Many other mammalian proteins that do not have sequence homologs but do have functional homologs can be used to c

32、omplement function in microbial cells.The functions of about half of the yeast and E. coli genes are known on the basis of amino acid sequence similarity with other proteins of known function.The knowledge of the function of microbial proteins will help in elucidating the function of many mammalian

33、proteins: “What is true for Escherichia coli is true for the eleohant, except more so” -Jacques Monod.Biological relevance of microbial screening systemsa. Antibacterial activitycompoundsInhibition zoneThese assays have been automated with robots under sterile environment in major Pharma.b. Growth/N

34、o growth assaysWith functional expression of homologous or heterologous targetsin microbial systems, rendering the cell dependent on the target expressed, a growth or no growth (of the microbe) type of screencan be developed.Example: S. cerevisiaebased screen for immunosuppressantsImmunosuppressants

35、, such as cyclosporin and FK506, inhibit T cell activation and have made tissue and organ transplantation a reality.Cyclosporin and FK506 bind proteins with peptidyl prolyl isomerase activity and forms a complex that inhibits the calcium-calmodulin phosphatase, calcineurin.In T cells and in yeast, f

36、ree calcineurin is essential for the activation of transcription factors.In the presence of these immunosuppressants, yeast dose not grow and divide.2022/7/1962c. Reporter-based assaysA target protein is coupled to a promoter (transcriptional factor)that in turn is coupled to a reporter protein like

37、 -galactosidase (半乳糖苷酶) , luciferase (荧光素酶), or chloramphenicol (氯霉素) acetyl transferase. Thus a target protein is engineered in the extracellular domain of the ToxR protein in E. coli. When compou-nds bind to the target protein, promote dimerization (二聚) of extracellular domain of hybrid ToxR prote

38、in, which activates the toxR promoter and consequently activates the expression of reporter and can be easily read in a plate reader.d. Yeast ExpressionAs yeast offers null background for human receptors, human GPCRs along with appropriate mammalian G-proteins can be expressed in yeast coupled to th

39、e pheromone signaling pathway(信息素传导途径) to screen for agonists and antagonists.GPCRs (G-protein coupled receptors): serotonin receptors, dopamine receptors, adrenergic receptors.The mating factor receptor in S. cerevisiae is called Ste2 and is similar in structure to mammalian GPCRs.Mammalian GPCR ca

40、n be used to replace Ste2, so that GPCR can signal through the mating factor signaling pathway when activated by the GPCR agonist.2022/7/1964e. Two-hybrid Yeast systemProtein-protein expression2、Mammalian Cell-Based AssaysCell-based assays differ from more traditional screening enzyme- or antibody-b

41、ased assays in that the use of live cells requires special considerations. a/ free of mycoplasma（支原体）; b/ cells from frozen stock should be viable without alteration in the growth curve; c/ target protein should be expressed at a high enough level in the cells; d/ little fluctuation in replicates .

42、Cell-based assays will take several days before initiation of the assays.The readouts of homogeneous format are radioactive, luminescence, or fluorescence.1). Radioactive AssaysCell-based radioactive homogeneous assays havebeen used for functional assays and receptor-ligandassays.Receptor binding as

43、saysReceptor binding assays with membrane receptorscan also be assays with whole cells, either adherentor suspension cells, with a radioligand.2022/7/1967GTP-S binding assaysG-protein activation can be assessed by measuring the agonist induced binding of a nonhydrolyzable GTP analog 35SGTP S to cell

44、s: SPA FlashPlates2022/7/1968Signal transduction assayscAMP: SPAThe SPA assay is based on competition between the cellular cAMP and exogeneously added tracer of 125I-cAMP. The radiolabeled cAMP binds to cAMP-specific antibody, which binds to the SPA bead coated with secondary antibody.The signal is

45、detected due to the close proximity of the radiolabel to the scintillant on the bead.2). Nonradioactive AssaysCyclic AMP assaysA high efficiency fluorescence polarization (HEFP)cAMP assay is a homogenous cAMP assay that can measure cAMP levels in whole cells and is based on competition between cAMP

46、produced in the cell andexogeneously added fluorescent cAMP as trancer (LJL BioSystems)Cell Incubated CellLysedDrugFluorescent trancercAMP-specificantibodyFP signalmeasurement2022/7/1971Ca2+ assaysFluorescent dye:Fura-2, Indo-1Fluo-3, Fluo-4,Rhod-23). Reporter-based AssaysMost of the transcription f

47、actors are modular, consistingof a DNA-binding domain and activation domain. Thesedomains can be interchanged between different factorsand still retain their functional properties.A reporter gene construct consists of an inducible trans-criptional control element driving the expression of a reporter

48、 gene.Reporter genes code for proteins that possess unique enzyme activities, and the activity assays are adaptable toHTS.2022/7/1973视黄酸受体(RAR)和视黄素X受体(RXR) 由3种不同的基因：,和编码，形成了RAR，RAR、RAR和RXR、RXR、RXR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

49、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

50、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)。RARRXRLBDDBDRARETTNPBLG268AGGTCA AGGTCA AGGTCADR1DR153DR1LacZReporter geneSRC-1：LXXLL ClampExperimental model of RAR-RXR heterodimers:RARE: biotinylated retinoic-acid-res

51、ponse element3、Miscellaneous AssaysLead compounds and compounds of interest for lead optimization are routinely tested for cytotoxicity, inhibition of cytochrome P450 isoenzymes (CYPs), compound permeability in Caco-2 cells, and specificityin other related assays.Profiling of the compounds at the ti

52、me of the lead optimization is very helpful for selecting compounds toin vivo studies.1) Cell proliferation and cytotoxicity assaysThe effect of compounds on the cell is generally assessed with nonspecific cytotoxicity assays. Cell viability testCellMTT(tetrazolium)Living cells reduce MTT to a highl

53、y coloredformazen salt and can be read in a platereader as an end point reading.MTT-relatively insoluble(MTS/XTT)2022/7/1978Alamar Blue Assays-is a widely used homogeneous cell proliferation/cytotoxic assay that can be performed in 96- or 384-well plates in HTS modeWhen Alamar Blue is added to eithe

54、r adherent or suspension cell culture, the dye becomes reduced to a highly fluorescent red form that can be read in a spectrophotometric or fluorescence reader. The intensity of color or fluorescence is proportional to the cell viability. Alamar Blue is nontoxic to cells and allows continuous monito

55、ring of cells at various periods of time.2) CYPsLead optimizationstudiesADME/PKPromoteEarly LeadsTo CompoundsAbsorptionDistributionMetabolismExcretionPharmacokinetics3) Chip TechnologiesMicrofabrication and microfluidies-based chip technologyis emerging and may replace HTS with further miniturizatio

56、nMicrochip technology has become a powerful technologyand is widely used in DNA analysis.DNA chip technologyWithin the department of Molecular Genetics, DNA chip technology is a core activity. This technology, which is also synonymous with DNA micro-array analysis, aims at taking a global view of gene activities, thus enabling the researcher to simultaneously follow the expression levels of thousands of genes in a single experiment.The purpose of this