Assay for HTS of Gi/Go-linked GPCRs: mGluR7 as Prot(RMI)

摘要 靶向与GTP结合蛋白偶联的受体的药物代表着良好的临床前景 跨越多种人类疾病的药物。在G蛋白偶联受体(GPCR)超家族中， 开发高通量筛选策略来识别药物调节作用一直是最具挑战性的 Gi/Go连接的受体。以前HTS检测这些受体的方法都是间接的、繁琐的， 而且昂贵，依赖于抑制药物刺激的cAMP积累或使用嵌合体 G蛋白。我们建议开发一种通过测量铊流量来筛选gi/o连接受体的策略。 通过G蛋白调节内向整流钾(K+)(GIRK)通道。使用此技术在 HeK细胞稳定表达GIRK 1和GIRK 2通道，我们已经用毒扁豆碱产生了初步数据 激动剂和拮抗剂，并观察到M2和M4对这些通道的剂量依赖性调节 有选择性的药物。使用384井板格式，我们已经生成了&gt；0.5的初步Z‘值，表明 这项测试是服从HTS的。我们计划使用这项技术来进行一个小规模的8000个化合物， 定向HTS筛选与代谢性谷氨酸受体7(MGluR7)相互作用的药物。这 基于其细胞定位和功能活性的受体在体外与GIRK偶联 预计将成为治疗神经和精神疾病的新靶点。最后，我们会 执行二次筛查以验证潜在的命中率，并确定mGluR7与其他 MGluRs。预计这些研究将为GI/GO相关受体的筛选开辟新的途径 并为mGluR7生成有价值的工具和药物线索。 版面总结 G蛋白偶联受体(GPCRs)是人类疾病中可获得的治疗靶点。在 对于GPCR家族，开发技术直接、省时、敏感和成本- 识别与GI/GO GTP结合蛋白偶联的药物靶向受体的有效方法。我们建议 开发和表征一种新的高通量筛选技术，用于通过Gi/o传递信号的受体 调节G蛋白调节的内向整流钾(K+)通道。利用代谢性 谷氨酸受体7(MGluR7)作为Gi/o偶联受体的初始原型，我们将筛选出一个小的、靶向的 为调节mGluR7的试剂开发新的工具和潜在的先导化合物的文库 哪些有限的药理药物是可用的，哪些代表了神经科的新药物靶点 以及精神疾病，如癫痫和精神分裂症。

Abstract Pharmacological agents targeting receptors coupled to GTP binding proteins represent promising clinical agents spanning a multitude of human diseases. Within the G-protein coupled receptor (GPCR) superfamily, it has been most challenging to develop high throughput screening strategies to identify drugs modulating Gi/Go-linked receptors. Previous HTS methods examining these receptors have been indirect, cumbersome, and expensive, relying upon either an inhibition of drug-stimulated cAMP accumulation or the use of chimeric G-proteins. We propose to develop a screening strategy for Gi/o-linked receptors by measuring thallium flux through G protein regulated Inwardly Rectifying Potassium (K+) (GIRK) channels. Using this technique in HEK cells stably expressing GIRK 1 and 2 channels, we have generated preliminary data using muscarinic agonists and antagonists and have observed dose-dependent regulation of these channels by M2 and M4 selective agents. Using a 384 well plate format, we have generated preliminary Z' values of >0.5, indicating that this assay is amenable to HTS. We plan to use this technique to perform a small scale, 8000 compound, directed HTS screen for drugs interacting with the metabotropic glutamate receptor 7 (mGluR7). This receptor, based upon its cellular location and functional activity, is known to couple to GIRK in in vitro systems and is predicted to serve as a novel target for neurological and psychiatric disorders. Finally, we will perform secondary screens to validate potential hits and determine specificity for mGluR7 versus other mGluRs. It is anticipated that these studies will open new avenues for Gi/Go-linked receptor screening as well as generate valuable tools and drug leads for mGluR7. Lay summary G-protein coupled receptors (GPCRs) represent accessible therapeutic targets in human disease. Within the GPCR family, it has been challenging to develop technically direct, time-efficient, sensitive, and cost- effective assays to identify drugs targeting receptors coupled to Gi/Go GTP binding proteins. We propose to develop and characterize a new high throughput screening technique for receptors that signal through Gi/o to regulate the G protein regulated Inwardly Rectifying Potassium (K+) channel. Using the metabotropic glutamate receptor 7 (mGluR7) as an initial prototype Gi/o-coupled receptor, we will screen a small, targeted library to develop new tools and potential lead compounds for agents modulating mGluR7, a protein for which limited pharmacological agents are available and which represents a novel drug target in neurological and psychiatric diseases such as epilepsy and schizophrenia.