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

基本信息

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
项目类别：
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

项目摘要

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）：有关药物信号传导途径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% 的处方药都以 GPCR 为靶点。这些蛋白质是许多药物发现计划的焦点，因为它们是癌症、心脏病、糖尿病、中枢神经系统疾病、炎症和疼痛等多个领域的治疗靶点。许多 GPCR 信号通路的关键组成部分是第二信使二酰甘油 (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化、稳健的 DAG 检测方法。该提案解决的挑战是生产基因编码的稳健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 生产极其坚固的基因编码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，但由于信噪比差和响应动态范围低，其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输送到活细胞中。这些活细胞测定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。