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

摘要：靶向受体与GTP结合蛋白偶联的药理药物是跨越多种人类疾病的有前途的临床药物。在g蛋白偶联受体（GPCR）超家族中，开发高通量筛选策略来鉴定调节Gi/ go连接受体的药物是最具挑战性的。以前检测这些受体的HTS方法是间接的，繁琐的，昂贵的，依赖于抑制药物刺激的cAMP积累或使用嵌合g蛋白。我们建议通过测量铊通过G蛋白调节的内向整流钾（K+） （GIRK）通道的通量来筛选Gi/o连接受体。在稳定表达girk1和2通道的HEK细胞中使用该技术，我们获得了使用毒蕈碱激动剂和拮抗剂的初步数据，并观察到M2和m4选择性药物对这些通道的剂量依赖性调节。使用384孔板格式，我们生成了初步的Z'值>0.5，表明该分析适用于HTS。我们计划利用这项技术进行小规模的8000个化合物的定向HTS筛选，以寻找与代谢性谷氨酸受体7 （mGluR7）相互作用的药物。这种受体，基于其细胞位置和功能活性，已知在体外系统中与GIRK偶联，并被预测为神经和精神疾病的新靶点。最后，我们将进行二次筛选，以验证潜在的命中，并确定mGluR7与其他mGluRs的特异性。预计这些研究将为Gi/ go连接受体筛选开辟新的途径，并为mGluR7提供有价值的工具和药物先导。概述：g蛋白偶联受体（gpcr）是人类疾病中可获得的治疗靶点。在GPCR家族中，开发技术直接、时间高效、敏感和成本有效的检测方法来识别与Gi/Go GTP结合蛋白偶联的靶向受体的药物一直是一项挑战。我们建议开发并表征一种新的高通量筛选技术，用于通过Gi/o信号调节G蛋白调节的内向纠偏钾（K+）通道的受体。利用代谢性谷氨酸受体7 （mGluR7）作为初始原型Gi/o偶联受体，我们将筛选一个小的靶向文库，以开发新的工具和潜在的先导化合物来调节mGluR7。mGluR7是一种有限的药物可用的蛋白质，代表了神经和精神疾病（如癫痫和精神分裂症）的新药物靶点。

DESCRIPTION (provided by applicant): 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 M4selective 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.