Fast and Accurate Tools for Measuring Fluorescence in Living Cells

描述（由申请人提供）：关于药物信号通路中第二信使状态的特定信息是理解药物在细胞内机制的关键。最大的一类药物靶点是g蛋白偶联受体（GPCR），超过40%的处方药以GPCR为靶点。这些蛋白质是许多药物发现项目的重点，因为它们是多种领域的治疗靶点，包括癌症、心脏病、糖尿病、中枢神经系统疾病、炎症和疼痛。许多GPCR信号通路的关键组成部分是第二信使二酰基甘油（DAG），然而，目前还没有可用于药物筛选或基础研究应用的商业上可靠的DAG测定方法。该提案解决的挑战是生产遗传编码健壮且易于使用的活细胞检测产品，可用于研究和筛选GPCR激活。该项目的第一阶段表明，为DAG生产极其健壮的、基因编码的荧光传感器是可行的。这一发现意义重大，因为尽管遗传编码分析具有巨大的优势，但由于信号噪声差和反应的动态范围低，对药物发现的影响有限。在第二阶段，将对第一阶段开发的原型传感器进行优化和包装，以便输送到活细胞中。这些活细胞试验将在严格的高通量筛选条件下进行验证，以确定高通量和体内筛选应用的适用性。

DESCRIPTION (provided by applicant): Specific information about the state of second messengers in drug signaling pathways is key to understanding the mechanism of a drug within the cell. The largest class of drug targets is the G-Protein Coupled Receptor (GPCR) and over 40% of all prescription pharmaceuticals target a GPCR. These proteins are the focus of many drug discovery programs because they are therapeutic targets in a variety of areas including cancer, cardiac disease, diabetes, central nervous system disorders, inflammation, and pain. A critical component of many GPCR signaling pathways is the second messenger, diacylglycerol (DAG), however, there is no commercially available, robust assay for DAG that can be used in either drug screening or basic research applications. The challenge addressed by this proposal is to produce genetically-encoded robust and easy-to-use live cell assay products that can be used to study and to screen for GPCR activation. Phase I of this project demonstrated that it is feasible to produce extremely robust, genetically encoded, fluorescent sensors for DAG. This discovery is significant because despite their great advantages, the impact of genetically encoded assays on drug discovery has been limited because of poor signal to noise and low dynamic range of the response. In Phase II, the prototype sensors developed in Phase 1 will be optimized and packaged for delivery into living cells. These live cell assays will be validated under rigorous high throughput screening conditions to establish suitability for high throughput and in-vivo screening applications.