COMPLEMENTATION ASSAY FOR G PROTEIN LINKED RECEPTORS

G蛋白连接受体（GPRs）是一个完整的膜结合蛋白家族，参与介导神经递质、激素和生长因子的生物学作用。它们是药物开发的主要靶标，并且大量临床使用的药物与GPR结合。人类基因组计划的最新进展已经揭示了数百个（如果不是数千个的话）新的孤儿GPR。这些孤儿受体具有几乎无限的新药开发潜力，并在制药工业中产生了巨大的兴趣，将其用作新的治疗靶点。然而，选择性地与孤儿受体相互作用的药物的发现受到大多数GPR没有已知的内源性配体并且因此不能在配体结合测定中检测的法案的阻碍。GPR偶联G蛋白，并且GPR的激动剂刺激促进GTP与G蛋白的结合。因此，GTP与G蛋白结合的测量可用作配体与GPR和孤儿受体结合的功能筛选。DiscoveRx是一家新兴的生物技术公司，将开发一种简单，快速和灵敏的检测方法，用于激动剂刺激GTP γ S与G蛋白的结合。该检测采用CEDIA技术，这是一种基于酶片段体外互补的蛋白质-配体相互作用测量的均相方法。活性酶的形成通过G蛋白与GTP γ S-酶片段缀合物的结合来调节;形成的酶的量可以在溶液中使用显色或荧光底物来测定，而无需分离或洗涤步骤。本提案的目标是开发一种新的、易于使用的用于高通量筛选与GPR结合的配体的测定法。作为原理证明，DiscoveRx将开发CEDIA测定法来测量激动剂与克隆的μ阿片受体的结合。在本申请中，我们将优化用于检测μ受体的阿片样物质刺激的CEDIA测定的条件。这些实验将是未来研究（II期应用）的基础，以确定DiscoveRx技术测量药物与GPR结合的普遍适用性，并优化GPR和孤儿受体高通量筛选的测定。拟定商业应用：本提案中开发的DiscoveRx检测技术将通过向拥有GPR药物发现计划的公司（包括大型制药和生物技术公司）提供基于该技术的CEDIA检测（试剂盒和定制检测）和服务进行商业化。DiscoveRx将与这些公司合作，以优化分析，就像Aurora Biosciences许可和优化其高通量筛选分析与制药公司。这些分析的估计市场将在数百万美元。

G protein linked receptors (GPRs) are a family of integral membrane bound proteins involved in mediating the biological actions of neurotransmitters, hormones and growth actors. They are major targets for pharmaceutical drug development, and a large number of clinically used drugs bind to GPRs. The recent advances of the Human Genome Project have revealed hundreds if not thousands of novel orphan GPRs. These orphan receptors have an almost unlimited potential for new drug development and have generated substantial interest in the pharmaceutical industry for their use as new therapeutic targets. However, discovery of drugs that selectively interact with orphan receptors is hindered by the Act that most GPRs have no known endogenous ligands and therefore can not be detected in ligand binding assays. GPRs couple to G proteins, and agonist stimulation of GPRs promotes the binding of GTP to G proteins. Therefore, measurement of GTP binding to G proteins can be employed as a functional screen for ligand binding to GPRs and orphan receptors. DiscoveRx is a start up biotechnology company that will develop a simple, rapid and sensitive assay for agonist stimulation of the binding of GTPgammaS to G proteins. The assay employs CEDIA technology, which is a homogeneous method for measurement of protein-ligand interactions based on in vitro complementation of enzyme fragments. Formation of active enzyme is regulated by binding of the G protein to a GTPgammaS-enzyme fragment conjugate; the amount of enzyme formed can be determined in solution, without separation or wash steps, using chromogenic or fluorogenic substrates. The goal of this proposal is to develop a novel, easy to use assay for high throughput screening of ligand binding to GPRs. As a proof of principle, DiscoveRx will develop a CEDIA assay to measure the binding of agonists to the cloned mu opioid receptor. In this application, we will optimize the conditions of the CEDIA assay for the detection of opioid stimulation of the mu receptor. These experiments will be the basis of future studies (Phase II application) to determine the general applicability of the DiscoveRx technology to measure drug binding to GPRs and to optimize the assay for high throughput screening of GPRs and orphan receptors. PROPOSED COMMERCIAL APPLICATION: The DiscoveRx assay technology developed in this proposal will be commercialized by providing CEDIA assays (both as kits and as custom assays) and services based on this technology to companies with GPR drug discovery programs, including major pharmaceutical and biotech companies. DiscoveRx will work together with those companies to optimize the assay, much like Aurora Biosciences licenses and optimizes its high throughput screening assays with pharmaceutical companies. The estimated market for these assays will be in the millions of dollars.