COMPLEMENTATION ASSAY FOR G PROTEIN LINKED RECEPTORS

G蛋白连接受体(GPR)是一个完整的膜结合蛋白家族，参与调节神经递质、激素和生长因子的生物学作用。它们是药物开发的主要靶点，大量临床使用的药物与GPRS绑定。人类基因组计划的最新进展揭示了成百上千个新的孤儿GPR。这些孤儿受体具有几乎无限的新药开发潜力，并因其作为新的治疗靶点而引起制药行业的极大兴趣。然而，大多数GPR没有已知的内源性配体，因此不能在配体结合分析中检测到，这一法案阻碍了选择性与孤儿受体相互作用的药物的发现。GPRS与G蛋白偶联，激动剂的刺激促进GTP与G蛋白的结合。因此，GTP与G蛋白结合的测定可作为GPR和孤儿受体配体结合的功能筛选。DiscoveRx是一家初创的生物技术公司，将开发一种简单、快速和敏感的测试方法，用于激动剂刺激GTPGammaS与G蛋白的结合。该分析采用CEDIA技术，这是一种基于酶片段体外互补的蛋白质-配体相互作用的均相方法。活性酶的形成是通过G蛋白与GTP-GammaS-酶片段结合物的结合来调节的；形成的酶的量可以在溶液中使用显色或荧光底物来测定，无需分离或洗涤步骤。这项建议的目标是开发一种新的、易于使用的方法，用于高通量筛选与GPR结合的配体。作为原则的证明，DiscoveRx将开发一种CEDIA方法来测量激动剂与克隆的u阿片受体的结合。在这一应用中，我们将优化CEDIA法检测阿片类药物对Mu受体刺激的条件。这些实验将成为未来研究(第二阶段应用)的基础，以确定DiscoveRx技术测量药物与GPR结合的普遍适用性，并优化高通量筛选GPR和孤儿受体的分析方法。拟议的商业应用：本提案中开发的DiscoveRx分析技术将通过向有GPR药物发现计划的公司(包括主要的制药和生物技术公司)提供CEDIA分析(作为试剂盒和定制分析)和基于该技术的服务而实现商业化。DiscoveRx将与这些公司合作优化检测，就像Aurora Biosciences的许可证一样，并与制药公司一起优化其高通量筛查检测。据估计，这些检测方法的市场价值将高达数百万美元。

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.