A NOVEL cAMP ASSAY FOR G PROTEIN-COUPLED RECEPTORS

描述(申请人提供)：所有人类细胞都受细胞外信号的调节，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了G蛋白偶联受体(GPCRs)超家族，作用于GPCRs的药物被广泛用于治疗各种人类疾病，包括帕金森氏症等神经系统疾病。GPCR是目前药物发现计划中最大的单一靶点，在大约200个已知功能的GPCRs中，由于技术限制，目前只有很小一部分GPCR用于药物发现，这使得许多GPCR成为开发新疗法的假定新靶点。他们中的大多数通过第二信使营地发出信号。 ATTO Bioscience开发了一种新的cAMP检测方法，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第一阶段拨款的支持下，我们建立了稳定的细胞系，并优化了用钙离子和膜电位染料检测CNG通道活性的实验条件。CNG-cAMP技术适用于多种检测平台，包括FLIPR和台式荧光平板阅读器。这是唯一的商业上可用的高通量筛选(HTS)分析系统，能够以时间分辨率检测受体激活。这项技术已经被授权给制药公司进行药物发现，验证了这项技术及其潜在市场。 我们的目标是开发全系列产品，使CNG-cAMP生物传感器技术能够商业化。它包括开发更多的Gs和Gi偶联的受体细胞系，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，还将使研究人员能够以一种以前不可能实现的新方式来检查受体激活和信号转导。 具体目标是：(1)建立、评估和生产另外20个用于药物发现和受体生物学研究的Gs偶联受体细胞系；(2)开发和生产40个GI偶联受体细胞系；(3)将CNG传感器引入10个不同的细胞系并将其开发为适销对路的产品；(4)设计、合成和评估新的膜潜在染料，并生产足够的试剂用于30,000,000次试验；(5)鉴定和测试新的猝灭剂，并开发2种用于CNG-cAMP检测的试剂盒；(6)建立GPCR阵列检测方法。

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.