HTS Cell Based EFC Assays for GPCR Drug Discovery(RMI)

描述（由申请人提供）：GPCR介导体内神经递质和激素的许多细胞作用，以及制药行业销售的几乎一半药物的治疗效果，并且已经成为药物发现的重要分子靶标。人类基因组测序已经揭示了多达800个GPCR基因，其中大多数是具有未知配体的孤儿。这些孤儿GPCR代表了用于发现新疗法的大量独特分子靶标。我们建议开发一种新的方法HTS的药物对孤儿GPCR采用的基本发现，激动剂结合GPCR的原因B-抑制蛋白从胞质到GPCR的易位。B抑制蛋白运输通常通过共聚焦显微镜来测量，其是低通量的、昂贵的并且不容易适用于HTS。我们建议开发一种简单的B-抑制蛋白运输试验，很容易适用于HTS使用我们的酶片段互补（EFC）技术，可以检测蛋白质的细胞内易位。该检测方法基于一种极其灵敏的B半乳糖苷酶（B-gal）互补技术，该技术利用了2个基因工程改造的E. coli B-gal。较大的片段，酶受体（EA），含有氨基末端附近的缺失，而较小的片段，ProLabel，含有EA缺失的氨基末端序列。单独，EA是不活动的。然而，在体外，它可以自发地与Pro标记重组，形成一种活性酶，催化形成一种发光产物，可以检测光度。该资助的目的是使用我们的EFC技术来测量响应于GPCR激活的B-抑制蛋白易位，作为孤儿GPCR和一般GPCR的高灵敏度药物发现测定。该技术还可用于鉴定GPCR的变构调节剂，其是一类新型药物，其改变GPCR活化的敏感性和程度而不直接与配体结合结构域相互作用。已知的变构调节剂是离子和G蛋白，其影响GPCR对激动剂和药物如MPEP的亲和力，所述激动剂和药物拮抗经由一些代谢营养型受体的传递而不影响激动剂结合。变构调节剂可以具有新的治疗用途，因为它们主要在受体被刺激时起作用，从而调节受体的递质和激动剂活化。在这项资助中，我们将测试开发的GPCR EFC测定是否可以用作鉴定GPCR变构调节剂的灵敏测定，以提供GPCR药物发现的全新途径。

DESCRIPTION (provided by applicant): GPCRs mediate many of the cellular actions of neurotransmitters and hormones in the body as well as the therapeutic effects of almost half of the drugs sold by the pharmaceutical industry and have been important molecular targets for drug discovery. The sequencing of the Human Genome has revealed as many as 800 GPCR genes, most of which are orphans with not known ligands. These orphan GPCRs represent a large pool of unique molecular targets for the discovery of new therapeutics. We propose to develop a novel approach for HTS of drugs against orphan GPCRs employing the basic finding that agonist binding to GPCRs causes the translocation of B-arrestin from the cytosol to the GPCR. B arrestin trafficking is usually measured by confocal microscopy which is low throughput, expensive and not easily amenable for HTS. We propose to develop a simple B-arrestin trafficking assay that is easily amenable for HTS using our enzyme fragment complementation (EFC) technology that can detect the intracellular translocation of proteins. The assay is based on an extremely sensitive B galactosidase (B-gal) complementation technology that utilizes 2 genetically engineered fragments of E. coli B-gal. The larger fragment, Enzyme Acceptor (EA), contains a deletion near the amino terminus, while the smaller fragment, ProLabel, contains the amino-terminal sequence missing from EA. Alone, EA is inactive. However, in vitro it can spontaneously recombine with Pro-label to form an active enzyme that catalyzes the formation of a luminescent product that can be detected photometrically. The objective of this grant will be to use our EFC technology to measure B-arrestin translocation in response to GPCR activation as a highly sensitive drug discovery assay for orphan GPCRs and GPCRs in general. This technology could also be used to identify allosteric modulators of GPCRs which are a novel class of drugs that modify sensitivity and extent of GPCR activation without directly interacting with the ligand binding domains. Known allosteric modulators are ions and G proteins, which affect the affinity of GPCRs for agonists and drugs such as MPEP that antagonize transmission via some metabotrophic receptors without affecting agonist binding. Allosteric modulators can have novel therapeutic uses since they primarily act when the receptor is stimulated, thereby tempering transmitter and agonist activation of the receptor. In this grant, we will test whether the GPCR EFC assay developed can be employed as a sensitive assay to identify GPCR allosteric modulators to provide a totally novel avenue of GPCR drug discovery.