Fast and Accurate Tools for Measuring Fluorescence in Living Cells

用于测量活细胞荧光的快速准确的工具

• 批准号: 8648063
• 负责人: THOMAS E HUGHES
• 金额: $ 58.55万
• 依托单位: MONTANA MOLECULAR, LLC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-15 至 2015-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8648063
• 关键词: 1,2-diacylglycerol; Acetylcholine; Address; Agonist; Area; Basic Science; Biological; Biological Assay; Cell Line; Cells; Central Nervous System Diseases; Cholinergic Receptors; Color; Deposition; Diabetes Mellitus; Diglycerides; Drug Targeting; Enzymes; Fluorescence; Fluorescence Resonance Energy Transfer; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genes; Goals; Green Fluorescent Proteins; Health; Heart; Heart Diseases; Human; Inflammation; Length; Life; Malignant Neoplasms; Maltose; Mammalian Cell; Measurement; Measures; Muscarinic M1 Receptor; Muscarinics; Neurons; Noise; Pain; Pathway interactions; Pattern; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Positioning Attribute; Preclinical Drug Evaluation; Process; Production; Proteins; Reader; Receptor Activation; Reproducibility; Second Messenger Systems; Signal Pathway; Signal Transduction; Site; Structure; Subfamily lentivirinae; System; Testing; Transgenic Mice; Treatment outcome; Variant; Virus; base; cell type; cost; drug discovery; expression vector; heart cell; high throughput screening; improved; in vivo; meetings; programs; prototype; public health relevance; receptor; red fluorescent protein; response; screening; second messenger; sensor; stable cell line; statistics; therapeutic target; tool

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.

描述(由申请人提供)：关于药物信号通路中第二信使状态的具体信息是了解药物在细胞内的机制的关键。最大的一类药物靶点是G蛋白偶联受体(GPCR)，所有处方药中超过40%的靶点是GPCRa。这些蛋白质是许多药物发现计划的焦点，因为它们是各种领域的治疗靶点，包括癌症、心脏病、糖尿病、中枢神经系统疾病、炎症和疼痛。第二信使二酰甘油(DAG)是许多GPCR信号通路的关键成分，然而，目前还没有一种可商业化的、可靠的DAG检测方法可用于药物筛选或基础研究应用。这项提议所解决的挑战是生产基因编码的、健壮的和易于使用的活细胞分析产品，这些产品可以用于研究和筛选GPCR激活。该项目的第一阶段证明了为DAG生产极其坚固的、遗传编码的荧光传感器是可行的。这一发现意义重大，因为尽管基因编码分析具有巨大的优势，但由于信噪比低和反应的动态范围低，对药物发现的影响有限。在第二阶段，在第一阶段开发的原型传感器将进行优化和包装，以便输送到活细胞中。这些活细胞分析将在严格的高通量筛选条件下进行验证，以确定是否适合高通量和体内筛选应用。