Detection of GPCR Function in Cell-free Nanoscale System

无细胞纳米系统中GPCR功能的检测

• 批准号: 6936101
• 负责人: Benjamin Jacob Doranz
• 金额: $ 19.19万
• 依托单位: INTEGRAL MOLECULAR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6936101
• 关键词: bioassay; bioengineering /biomedical engineering; biosensor device; biotechnology; cell component structure /function; cell free system; cell surface receptors; drug detection; fluorescence resonance energy transfer; high throughput technology; method development; microarray technology; nanotechnology; protein protein interaction; protein structure function; receptor binding; receptor expression; sensory signal detection

DESCRIPTION (provided by applicant): The G protein-coupled receptor (GPCR) CXCR4 is a determinant of the pathogenesis of HIV-1 infection and breast cancer, but efforts to develop drugs that modify its function have been unsuccessful. Traditional assays that screen GPCR function mostly use live cells or cell membrane fragments, formats that are limited by poor receptor purity, stringent environmental requirements, unacceptable variability, and an inability to be miniaturized, prohibiting their application to more versatile micro- and nano-scale detection technologies. Furthermore, traditional assays for GPCR activation often involve detection of downstream signaling events such as calcium release which are not stimulated by a number of important GPCRs and G proteins. A nanometer-scale, cell-free assay for GPCR activation that can be applied to microfluidic drug-screening devices could have a major impact on the discovery of drugs to difficult GPCR targets, and this proposal is designed to produce such a system. Our approach has three major advantages compared with existing assays: 1) simplicity, 2) the ability to be miniaturized to the nanometer scale, and 3) the ability to detect diverse and difficult GPCRs that conventional systems fail to detect. The product will facilitate high throughput screening of GPCRs, orphan ligand-receptor pairing, and the development of therapeutics that treat AIDS and breast cancer.

描述（由申请人提供）： G蛋白偶联受体（GPCR）CXCR4是HIV-1感染和乳腺癌发病机理的决定因素，但是开发改变其功能的药物的努力未成功。传统的测定法认为屏幕GPCR功能主要使用活细胞或细胞膜片段，这些格式受受体纯度差，严格的环境需求，不可接受的可变性以及无法小型化的限制，禁止其应用于更广泛的微型和纳米尺度检测技术。此外，传统的GPCR激活测定法通常涉及检测下游信号事件，例如钙释放，而钙释放并未受到许多重要的GPCR和G蛋白的刺激。可以应用于微流体药物筛查装置的纳米尺度，无细胞的GPCR激活测定法可能会对难以发现的困难GPCR靶标有重大影响，并且该建议旨在生产此类系统。与现有测定相比，我们的方法具有三个主要优势：1）简单性，2）将小型化到纳米量表的能力，以及3）检测常规系统无法检测到的多样化和困难的GPCR的能力。该产品将促进GPCR，孤儿配对配对的高吞吐量筛查以及治疗辅助和乳腺癌的治疗剂的发展。