中國(guó)藥物篩選平臺(tái)（ScreeningPlatforms）

1、中國(guó)藥物篩選平臺(tái)（Screening Platforms）藥物篩選在新藥發(fā)現(xiàn)中的地位藥物篩選的常見(jiàn)重要方法 重要疾病治療藥物篩選方法舉例展望 虛擬篩選技術(shù) （計(jì)算機(jī)科學(xué)） 生物物理化學(xué)技術(shù)、組合化學(xué) 分子細(xì)胞生物學(xué) 天然產(chǎn)物化學(xué)（中草藥資源） （后基因組時(shí)代藥物篩選新模式）Biological Target SelectionAssay ReagentsAssay DevelopmentAnd OptimizationAssay TransferAssessmentScreen DevelopmentAnd ValidationScreen AutomationAnd Optimization

2、Screening Compound DeckLead GenerationScreen ParadigmHTS, uHTSScreen PlatformsIII、篩選平臺(tái)Being familiar with various screen platforms willbe in an advantageous position to evaluate the bestassay for the target screening with the identificationof a target. (靶標(biāo)）Understanding various screen platforms will

3、 help in arriving at the best screens with the available equipment and reagents. （設(shè)備和試劑）TargetsReagents and plate readers availability Assay Design1、 Assay formats20世紀(jì)70年代：低通量和單一試管篩選方法；現(xiàn)在:(1)多孔板篩選 （96、384、1536，3456，9600 孔板篩選技術(shù)相繼出現(xiàn)）(2) 平板讀數(shù)（plate readers) 技術(shù)：熒光、發(fā)光、閃爍等 檢測(cè)技術(shù)的發(fā)展。 Stackers holding severa

4、l plates (10-40 plates).Biacore 3000，96生物分子相互作用分析系統(tǒng) Biacore S51，38496 2. Assay techniques依據(jù)：激活或抑制、 激動(dòng)劑(activator)、抑制劑 (inhibitor)方法:（1）體外篩選（in vitro)（酶活、受體結(jié)合） （cell-free）（2）細(xì)胞水平（cell-based)（Heterogeneous & Homogeneous types) 3、體外（in vitro）篩選 （cell free）采用系統(tǒng)簡(jiǎn)單或復(fù)雜（酶反應(yīng)、蛋白蛋白相互作用、膜受體配體結(jié)合、可溶性受體配體結(jié)合檢測(cè)實(shí)驗(yàn)）特點(diǎn)

5、 a 、被篩化合物易于直接作用于靶標(biāo)；b、目標(biāo)化合物作用靶標(biāo)明確；c、明確的作用機(jī)理；d、易于發(fā)展便宜易得的靶標(biāo)模型；e、適應(yīng)新技術(shù)的發(fā)展，易于自動(dòng)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多步篩選方法 multiple additions/incubations/washings/transfers/filtrations/readings of the signal特點(diǎn)Labor intensive, complicated step, hard to automate（1）Nonradioactiv

7、e Heterogeneous AssaysEnzyme immunoassays (widely used in vitro assays)ELISA （Enzyme Linked Immunosorbent Assay）酶聯(lián)免疫吸附試驗(yàn)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 方法藥物篩選原理Read !EGFR 抑制劑篩選ELISA試劑盒（2）Radioactive Heterogeneous AssaysVery commonly used, highly sensitive and robustdespite handling hazards and radioactive w

11、aste generation分離放射性產(chǎn)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 （磷酸纖維素）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.抗原對(duì)其抗體進(jìn)行免疫結(jié)合進(jìn)行的分析通常的抗原是指人體內(nèi)存在的激素，酶，小分子和多肽，特異蛋白等。對(duì)于其它動(dòng)物，即異體物質(zhì)，一旦把這些物質(zhì)引入動(dòng)物體內(nèi)，其免疫系統(tǒng)就會(huì)作出反應(yīng)，產(chǎn)生出專一結(jié)合抗原的抗體（即免疫球蛋白)?？乖涂贵w的反應(yīng)是一一對(duì)應(yīng)的，高度精密專一的。 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) (親近閃爍檢測(cè)) 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-羥

17、色胺腎上腺素生長(zhǎng)激素抑制素表皮生長(zhǎng)因子血管緊張素（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 (比色測(cè)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順十八碳四烯酸max (410 nm)PPARLigandCPAPPARmax (410 nm)2022/7/19372022/7/1938Fluorescence quench assays (CypA + Ligand)隨著DDDC838濃度增大，CypA熒光值下降，實(shí)驗(yàn)中，CypA濃度保持為4M，其中化合物DDDC838濃度：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) (熒光共振能量傳遞) assays (iii) Homogeneous time-resolved fluorescence (HTRF)/ Time-resolved FRET (iv) Fluorescence correlation spectroscopy (FCS) （熒光相干光譜學(xué)）(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 (熒光素酶), 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(信息素傳導(dǎo)途徑) 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（支原體）; 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視黃酸受體(RAR)和視黃素X受體(RXR) 由3種不同的基因：,和編碼，形成了RAR，RAR、RAR和RXR、RXR、RXR等多種類型的受體。受體的結(jié)構(gòu)由5個(gè)亞區(qū)組成，其中高度保守的DNA結(jié)合區(qū)(DBD)和低度保守的配體結(jié)合區(qū)(LBD)發(fā)揮重要的作用。在DBD結(jié)構(gòu)中，含有2個(gè)半胱氨酸豐富的鋅指，它與受體識(shí)別特異的DNA序列、受體形

49、成二聚體有關(guān)。在LBD結(jié)構(gòu)中，含有大量的疏水氨基酸，它與配體的連接、轉(zhuǎn)錄的抑制與激活有關(guān)?？梢?jiàn)，受體上不同的亞區(qū)行使不同的功能，由此構(gòu)成受體的復(fù)雜性和功能的多樣性。RAR和RXR屬于類固醇和甲狀腺激素受體超家族成員，它們作為配體激活的轉(zhuǎn)錄因子，結(jié)合到靶基因的特定應(yīng)答序列(DNA)上，調(diào)節(jié)基因的轉(zhuǎn)錄表達(dá)。這個(gè)特定的DNA序列稱為激素應(yīng)答元件，視黃酸應(yīng)答元件(RARE)是其中的一個(gè)典型代表，受體與RARE的結(jié)合則是維生素A信號(hào)傳導(dǎo)的關(guān)鍵。研究表明，RAR本身不能形成同源二聚體而結(jié)合到RARE上，它必須要有輔助性蛋白的幫助，才能進(jìn)行有效的DNA結(jié)合。1992年發(fā)現(xiàn)RXR就是這種輔助性蛋白，RXR通過(guò)

50、與RAR形成異源二聚體(RAR/RXR)，增加RAR與RARE的親和力，由此加強(qiáng)RAR的轉(zhuǎn)錄活性和對(duì)配體的敏感性。此外，RXR在其配體如9-cisRA的存在下，還可以形成同源二聚體(RXR/RXR)而結(jié)合到DNA序列上，由此介導(dǎo)維生素A不同的應(yīng)答途徑(如圖)。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