《筛选平台85中国药科大学药物筛选幻灯片》由会员分享，可在线阅读，更多相关《筛选平台85中国药科大学药物筛选幻灯片（85页珍藏版）》请在金锄头文库上搜索。
1、第二章、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 Selection,Assay Reagents,Assay Development And Optimization,Assay Transfer,Assessment,Screen Development And Validation,Screen Automation An
2、d Optimization,Screening Compound Deck,Lead Generation,Screen Paradigm,HTS, uHTS,Screen Platforms,III、筛选平台,Being familiar with various screen platforms will be in an advantageous position to evaluate the best assay for the target screening with the identification of a target. (靶标） Understanding vari
3、ous screen platforms will help in arriving at the best screens with the available equipment and reagents. （设备和试剂）,Targets,Reagents and plate readers availability,Assay Design,1、 Assay formats,20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
4、检测技术的发展。 Stackers holding several plates (10-40 plates).,Biacore 3000，96 生物分子相互作用分析系统,Biacore S51，384,96,2. 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
5、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。,In vitro cell-free biochemical assays,Homogeneous,Heterogeneous,Radioactive assays,SPA bead,SPA plate,Cell-free Biochemical Assays,Chromogenic assays,Non-radioactive assays,A
6、bsorbance assays,Fluorescence assays,Bead based assays,Radioactive assays,Non-radioactive assays,Filtration assays,Adsorption assays,Precipitation (沉淀) assays,Radioimmunoassays,ELISA assays,A. Heterogeneous Assays,多步筛选方法 multiple additions/incubations/washings/ transfers/filtrations/readings of the
7、signal,特点 Labor intensive, complicated step, hard to automate,（1）Nonradioactive Heterogeneous Assays,Enzyme immunoassays (widely used in vitro assays),ELISA （Enzyme Linked Immunosorbent Assay） 酶联免疫吸附试验,An ELISA plate,An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the first and mos
8、t basic test to determine if an individual is positive for a selected pathogen, such 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 Test,Negative ELISA Test,HIV antigen
9、s pre-coated onto an ELISA plate,Patient serum containing antibodies. If the patient is 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
10、human antibodies.,Chromogen or substrate which changes color when cleaved by the enzyme attached to the second antibody.,S,E,P,Antibody,Antibody-2,colour, fluorescence, chemiluminescence,Inhibitor Screening,S,Substrate,E,Enzyme,P,Product,ELISA 方法药物筛选原理,Read !,EGFR 抑制剂筛选 ELISA试剂盒,（2）Radioactive Heter
11、ogeneous Assays,Very commonly used, highly sensitive and robust despite handling hazards and radioactive waste generation,分离放射性产物的一般方法,Radioactive Product,Radioactive Substract,Glass-fiber filters,(filtration),Washing,Drying at rt.,Transferred into a vial,add scintillant,Counted in a scintillation c
12、ounter,a. Filtration Assays,b. Adsorption Assays,In protein kinase reactions the phosphorylated product (acidic) by ionic interaction 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 scintillati
13、on counter.,c. Precipitation Assays,In the traditional enzyme assays, the radiolabel from the substrate is transferred to a protein acceptor, and the radiolabeled product is isolated by precipitation with trichloroacetic acid (TCA, 三氯乙酸); the precipitate is collected by filtration and washing, the f
14、ilter is transferred into a viral,and the viral is counted after the addition of scintillant.,d. Radioimmunoassays(RIA),A classical 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（
15、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 step or in multisteps The signal is read in a plate reader,（1）Radioactive Homogeneous Assays,Based on scintillation proximity assay (SPA) (亲近闪烁检测) with either SPR be
16、ads or scintillant-coated plates.,Scintillation proximity assay (SPA), an innovative approach for high-throughput screening introduced in 1991, 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 stan
