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

描述（由申请人提供）：GPCRs介导体内许多神经递质和激素的细胞活动，以及制药行业销售的几乎一半药物的治疗效果，并且已成为药物发现的重要分子靶点。人类基因组测序已经揭示了多达800个GPCR基因，其中大部分是没有已知配体的孤儿基因。这些孤立的gpcr代表了一大批独特的分子靶点，可用于发现新的治疗方法。我们建议开发一种针对孤儿GPCR的药物HTS的新方法，利用激动剂与GPCR结合导致b -阻滞蛋白从细胞质转移到GPCR的基本发现。B逮捕贩运通常是用共聚焦显微镜测量的，这是低通量的，昂贵的，不容易适用于高通量显微镜。我们建议使用我们的酶片段互补（EFC）技术开发一种简单的b -阻滞蛋白运输检测方法，该方法易于适用于HTS，可以检测蛋白质的细胞内易位。该检测基于一种极其敏感的B-半乳糖苷酶（B-gal）互补技术，该技术利用了大肠杆菌B-gal的2个基因工程片段。较大的片段，酶受体（EA），包含一个氨基末端的缺失，而较小的片段，ProLabel，包含EA缺失的氨基末端序列。单独，EA是无活性的。然而，在体外，它可以自发地与Pro-label重组，形成一种活性酶，催化形成一种可以通过光度法检测到的发光产物。这项资助的目的是使用我们的EFC技术来测量响应GPCR激活的b -阻滞蛋白易位，作为孤儿GPCR和一般GPCR的高灵敏度药物发现试验。该技术还可用于鉴定GPCR的变构调节剂，这是一类新型药物，可以改变GPCR激活的敏感性和程度，而不直接与配体结合域相互作用。已知的变构调节剂是离子和G蛋白，它们影响gpcr对激动剂和药物（如MPEP）的亲和力，这些药物通过一些代谢受体拮抗传递，而不影响激动剂的结合。变构调节剂可以有新的治疗用途，因为它们主要在受体受到刺激时起作用，从而调节受体的递质和激动剂的激活。在这项资助中，我们将测试开发的GPCR EFC检测是否可以作为一种敏感的检测方法来识别GPCR变构调节剂，从而为GPCR药物发现提供一种全新的途径。

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.