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: 8791351
• 负责人: THOMAS E HUGHES
• 金额: $ 58.24万
• 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 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8791351
• 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; 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理解细胞内药物机制的关键。最大的一类药物靶点是G蛋白偶联受体（GPCR），超过40%的处方药靶向GPCR。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。许多GPCR信号传导途径的关键组分是第二信使，二酰基甘油（DAG），然而，没有可用于药物筛选或基础研究应用的DAG的市售的、稳健的测定。该提案所解决的挑战是生产可用于研究和筛选GPCR激活的基因编码的稳健且易于使用的活细胞测定产品。该项目的第一阶段表明，生产用于DAG的极其稳健的、遗传编码的荧光传感器是可行的。这一发现是重要的，因为尽管它们有很大的优势，但由于信噪比差和响应的动态范围低，遗传编码测定对药物发现的影响有限。在第二阶段，第一阶段开发的原型传感器将被优化和包装，以交付到活细胞中。将在严格的高通量筛选条件下验证这些活细胞测定，以确定高通量和体内筛选应用的适用性。