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

Gi/Go 连接的 GPCR 的 HTS 测定：mGluR7 作为 Prot(RMI)

• 批准号: 7407167
• 负责人: COLLEEN M NISWENDER
• 金额: $ 3.83万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7407167
• 关键词: Agonist; Biological Assay; Biology; Cell Line; Cells; Chemicals; Clinical; Co-Rax; Collection; Cyclic AMP; Data; Dose; Drug Delivery Systems; Epilepsy; Family; G-Protein-Coupled Receptors; GTP-Binding Proteins; Go Alpha Subunit; Heterotrimeric G Protein Subunit; In Vitro; Institutes; Ion Channel; Lead; Libraries; Link; Location; Measures; Mental disorders; Metabotropic Glutamate Receptors; Methods; Muscarinic Agonists; Muscarinic M1 Receptor; Neurologic; Output; Performance; Pertussis Toxin; Pharmaceutical Preparations; Potassium; Potassium Channel; Preclinical Drug Evaluation; Proteins; Range; Receptor Signaling; Regulation; Schizophrenia; Screening procedure; Series; Specificity; Standards of Weights and Measures; System; Techniques; Testing; Thallium; Time; Validation; abstracting; base; cost; cost effective; high throughput screening; human disease; in vivo; inward rectifier potassium channel; member; metabotropic glutamate receptor 7; novel; prototype; receptor; receptor coupling; research study; response; small molecule libraries; therapeutic target; tool

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.

摘要 靶向与GTP结合蛋白偶联的受体的药理学试剂代表了有前途的临床应用。 跨越多种人类疾病的病原体。在G蛋白偶联受体（GPCR）超家族中， 最具挑战性的是开发高通量筛选策略来鉴定调节肿瘤生长的药物， Gi/Go连接受体。以前检查这些受体的HTS方法是间接的，繁琐的， 并且昂贵，依赖于抑制药物刺激的cAMP积累或使用嵌合的 G蛋白我们建议通过测量铊通量来开发一种筛选Gi/o连接受体的策略 通过G蛋白调节的抑制整流钾（K+）（GIRK）通道。使用这种技术， HEK细胞稳定表达GIRK 1和2通道，我们已经产生了初步的数据， 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 选择剂。使用384孔板格式，我们已经产生了>0.5的初步Z'值，表明 该测定法适用于HTS。我们计划用这种技术进行一个小规模的，8000个化合物， 定向HTS筛选与代谢型谷氨酸受体7（mGluR 7）相互作用的药物。这 基于其细胞定位和功能活性，已知受体在体外与GIRK偶联 系统，并预计将作为一个新的目标，神经和精神疾病。最后我们将 进行二次筛选，以验证潜在的命中，并确定mGluR 7相对于其他 mGluRs。预计这些研究将为Gi/Go相关受体筛选开辟新的途径， 并为mGluR 7产生有价值的工具和药物线索。 简易摘要 G蛋白偶联受体（GPCR）代表了人类疾病中可获得的治疗靶点。内 GPCR家族中，开发技术上直接、省时、灵敏和成本低的 鉴定靶向与Gi/Go GTP结合蛋白偶联的受体的药物的有效测定。我们建议 开发并表征通过Gi/o信号传导的受体的新的高通量筛选技术 调节G蛋白调节的I型整流钾（K+）通道。使用代谢型 谷氨酸受体7（mGluR 7）作为初始原型Gi/o偶联受体，我们将筛选一个小的，靶向的 库开发新的工具和潜在的先导化合物，用于调节mGluR 7的药物，mGluR 7是一种蛋白质， 其是可用的有限的药理学试剂，并且其代表了神经病学中的新的药物靶点， 以及癫痫和精神分裂症等精神疾病。