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近一半药物的治疗效果，并且一直是药物发现的重要分子靶标。人类基因组测序揭示了多达800个GPCR基因，其中大部分是配体未知的孤儿基因。这些孤儿 GPCR 代表了大量用于发现新疗法的独特分子靶标。我们建议利用激动剂与 GPCR 结合导致 B-抑制蛋白从细胞质易位至 GPCR 的基本发现，开发一种针对孤儿 GPCR 的药物 HTS 的新方法。 B 视紫红质抑制蛋白的运输通常通过共聚焦显微镜来测量，但这种方法通量低、价格昂贵且不易适用于 HTS。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-抑制蛋白运输测定，该测定很容易适用于 HTS，该技术可以检测蛋白质的细胞内易位。该检测基于极其灵敏的 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)，包含氨基末端附近的缺失，而较小的片段，ProLabel，包含 EA 缺失的氨基末端序列。 EA 本身并不活跃。然而，在体外，它可以自发地与 Pro-label 重组，形成活性酶，催化形成可通过光度计检测的发光产物。这笔资助的目的是使用我们的 EFC 技术来测量 GPCR 激活反应中的 B-arrestin 易位，作为孤儿 GPCR 和一般 GPCR 的高度敏感的药物发现测定。该技术还可用于识别 GPCR 的变构调节剂，这是一类新型药物，可改变 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 MPEP 等药物的亲和力，这些激动剂和药物通过一些代谢营养受体拮抗传输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受到刺激时起作用，从而调节受体的递质和激动剂激活。在这笔资助中，我们将测试开发的 GPCR EFC 测定是否可以用作识别 GPCR 变构调节剂的灵敏测定，从而为 GPCR 药物发现提供全新的途径。

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.