A NOVEL cAMP ASSAY FOR G PROTEIN-COUPLED RECEPTORS

G 蛋白偶联受体的新型 Camp 测定

• 批准号: 6960033
• 负责人: Zhenjun Diwu
• 金额: $ 59.37万
• 依托单位: ANASPEC, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6960033
• 关键词: G protein; bioassay; biological transport; biotechnology; cell line; cell surface receptors; cyclic AMP; drug discovery /isolation; fluorescent dye /probe; high throughput technology; membrane channels; receptor expression; technology /technique development; time resolved data; transfection /expression vector

DESCRIPTION (provided by applicant): All human cells are regulated by extracellular signals that are transmitted through cell surface receptors. The vast majority of these receptors constitute a superfamily of G-protein coupled receptors (GPCRs), Drugs that act on GPCRs are widely used in therapies against a large variety of human diseases, including neurological diseases such as Parkinson's disease. GPCRs are the single largest target of current drug discovery programs yet, of the approximately 200 GPCR with known functions, only a very small fraction of them are currently utilized in drug discovery due to technology limitations, leaving many of them as putative new targets for the development of new therapeutics. The majority of them signal through second messenger cAMP. Atto Bioscience has developed a novel cAMP assay for a real-time detection of cAMP in living cells using a proprietary cyclic nucleotide-gated (CNG) channel mutant. With the support of a Phase 1 grant from NINDS, we created stable cell lines and optimized assay conditions with both Ca 2+and membrane potential dyes to detect activity of the CNG channels. The CNG-cAMP technology was adapted to multiple assay platforms including FLIPR and bench-top fluorescence plate readers. This is the only commercially available high throughput screen (HTS) assay system capable of detecting receptor activations with temporal resolution. The technology has been licensed for drug discovery to pharmaceutical companies, validating both the technology and its potential market. Our goal is to develop full lines of products to enable commercialization of CNG-cAMP biosensor technology. It includes the development of additional Gs- and Gi-coupled receptor cell lines, the development of CNG cell lines in different cell types, and the development of dye reagents for the technology. We believe that this cAMP biosensor technology will not only support drug discovery, but also allow researchers to examine receptor activation and signal transduction in a new way previously not possible. The specific aims are: (1) generate, evaluate and produce another 20 Gs-coupled receptor cell lines for drug discovery and receptor biological studies; (2) develop and produce 40 Gi-coupled receptor ceil lines; (3) introduce and evaluate CNG sensor into 10 different cell lines and develop them into marketable products; (4) design, synthesize, and evaluate new membrane potential dyes, and produce sufficient reagents for 30,000,000 tests; (5) identify and test new quenchers, and develop 2 reagent kits for CNG-cAMP assay; (6) develop assay for GPCR arrays.

描述（由申请人提供）：所有人类细胞均受细胞外信号调节，这些信号通过细胞表面受体传递。这些受体中的绝大多数构成G蛋白偶联受体（GPCR）的超家族。作用于GPCR的药物被广泛用于针对多种人类疾病的治疗，包括神经系统疾病如帕金森病。GPCR是目前药物发现计划的单一最大靶标，然而，在具有已知功能的大约200个GPCR中，由于技术限制，目前只有非常小的一部分用于药物发现，使其中许多成为开发新疗法的推定新靶标。其中大多数通过第二信使cAMP信号。 Atto Bioscience开发了一种新型cAMP测定法，用于使用专有的环核苷酸门控（CNG）通道突变体实时检测活细胞中的cAMP。在NINDS第一阶段资助的支持下，我们建立了稳定的细胞系，并优化了Ca 2+和膜电位染料检测CNG通道活性的测定条件。CNG-cAMP技术适用于多种测定平台，包括FLIPR和台式荧光板读数器。这是唯一一个能够以时间分辨率检测受体激活的市售高通量筛选（HTS）测定系统。该技术已被授权用于制药公司的药物发现，验证了该技术及其潜在市场。 我们的目标是开发全系列产品，使CNG-cAMP生物传感器技术商业化。它包括开发额外的Gs和Gi偶联受体细胞系，开发不同细胞类型的CNG细胞系，以及开发用于该技术的染料试剂。我们相信，这种cAMP生物传感器技术不仅将支持药物发现，而且还允许研究人员以一种以前不可能的新方式检查受体激活和信号转导。 具体目标是：（1）产生、评估和生产另外20个用于药物发现和受体生物学研究的G i偶联受体细胞系;（2）开发和生产40个G i偶联受体细胞系;（3）将CNG传感器引入和评估10个不同的细胞系中，并将它们开发成可销售的产品;（4）设计、合成、评价新型膜电位染料，生产出可满足3000万次检测的试剂;（5）鉴定、测试新型猝灭剂，开发2种CNG-cAMP检测试剂盒;（6）建立GPCR芯片检测方法。