An HTS-compatible Assay to Probe Muscarinic Receptor Modulation of the M-current

用于探测 M 电流的毒蕈碱受体调节的 HTS 兼容测定

• 批准号: 8102409
• 负责人: C DAVID WEAVER
• 金额: $ 15.58万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8102409
• 关键词: Acetylcholine; Address; Agonist; Atropine; Biological Assay; Cell Line; Cell membrane; Cells; Chemicals; Clinical Treatment; Clinical Trials; Collection; Coupled; Data; Detection; Development; Disease; Electrophysiology (science); Epilepsy; Family; Fluorescence; Fluorescent Dyes; GTP-Binding Proteins; Gated Ion Channel; Histamine; Ion Channel; KCNJ1 gene; Knowledge; Modality; Molecular; Muscarinic Acetylcholine Receptor; Muscarinic Agonists; Muscarinic Antagonists; Muscarinic M1 Receptor; Muscarinics; Names; Nervous System Physiology; Neurologic; Neurons; Pathologic Processes; Pathway interactions; Pharmacology; Phosphatidylinositol 4,5-Diphosphate; Phospholipase C; Physiological Processes; Play; Potassium Channel; Process; Proteins; Receptor Activation; Reporting; Research; Role; Running; Screening procedure; Signal Transduction; Staging; Structure of superior cervical ganglion; System; Technology; Tetracyclines; Thallium; Tissues; Transfection; Translating; Work; base; counterscreen; human CHRM1 protein; inhibitor/antagonist; interest; nervous system disorder; neuronal excitability; new technology; novel; novel therapeutics; promoter; protein phosphatase inhibitor-2; receptor; receptor coupling; release of sequestered calcium ion into cytoplasm; response; small molecule; stable cell line; tool; voltage

DESCRIPTION (provided by applicant): The M-current was first described approximately thirty years ago (Brown and Adams, 1980) and is now recognized as a key regulator of many neurological processes where it plays a dominant role in controlling excitability. In more recent years a number of small molecule modulators of the proteins that underlie the M- current (the Kv7 family of voltage-gated ion channels) have been reported. Both activators and inhibitors of the channel have been described and at least one activator, Retigabine, has progressed to late-stage clinical trials for the treatment of epilepsy. For years the mechanism that underlies the M-current's name (suppression of the current by agonist of Gq- coupled muscarinic acetylcholine receptors) remained mysterious. However, the work of Suh and Hille (2002) revealed that muscarinic receptor stimulated depletion of plasma membrane PIP2 was the likely mechanism governing muscarinic receptors suppression of the M-current. In subsequent years this mechanism of modulation of M-current has been of intense research interest. However, to date there have been no reports of easy-to-use; HTS-compatible assays to assess M-current modulation. Neither have there been any small- molecular tools reported that specifically target 7TM receptors' ability to modulate this critically important conductance. To address this problem we have initiated an effort to develop an HTS-compatible assay system and a suite of secondary assays to enable a screen focused on the discovery and characterization of small molecules that specifically modulate 7TM receptor suppression of M-current activity. Discovery of such tools will advance our understanding the role of 7TM receptors in modulating neuronal excitability via the M-current and may reveal novel therapeutic opportunities for Kv7 targets. Furthermore, with the growing appreciation of functional selectivity and context dependent pharmacology, there is an intense need for new 7TM receptor assay technologies that reflect known physiologically relevant effectors not presently addressed by standard assay technologies (e.g. intracellular calcium flux). The proposed M-current assay represents just such a novel assay system. In fact, though intracellular calcium flux is the most common cell-based functional assay technology for Gq-coupled muscarinic receptors, it appears that it is not the major signal transduction modality for muscarinic responses in some tissues including the neurons of the superior cervical ganglion (Hernandez et al, 2008) where muscarinic modulation of M-current is known to occur. Thus, not only does the proposed assay system represent an opportunity to discover novel and important small molecule probes, it also represents an important new mechanism for characterizing muscarinic receptor modulators. PUBLIC HEALTH RELEVANCE: The proposed research focuses on the development on novel technologies to enable the discovery of chemical modulators of the interactions between neuro-transmitter receptors and neuronal voltage-gated ion channels. These receptors and channels play key roles in nervous system function and are implicated in causing as well as treating numerous nervous system disorders. The proposed research will advance our knowledge regarding the role of these receptors and channels in normal and pathological processes with a focus on translating these into therapies for disease.

描述（由申请人提供）：大约30年前首次描述了M流动性（Brown and Adams，1980），现在被认为是许多神经系统过程中它在控制兴奋性中起主要作用的关键调节剂。近年来，已经报道了M-电流（电压门控离子通道的KV7家族）蛋白质的许多小分子调节剂。已经描述了该通道的激活因子和抑制剂，至少有一个激活剂Retigabine已发展为后期临床试验以治疗癫痫。多年来，基于M-电流名称的机制（抑制GQ耦合的毒蕈碱乙酰胆碱受体的电流）仍然是神秘的。然而，Suh和Hille（2002）的工作表明，毒蕈碱受体刺激了质膜PIP2的耗竭，这可能是管理M-毒品受体抑制M-电流的机制。在随后的几年中，这种调节M-电流的机制具有强烈的研究兴趣。但是，到目前为止，还没有易于使用的报道。与HTS兼容的测定法评估M电流调制。没有任何小分子工具报告，这些工具是针对7TM受体调节这种至关重要的电导能力的能力。为了解决这个问题，我们开始努力开发与HTS兼容的测定系统和一系列次级测定，以使筛选专注于发现和表征的小分子，这些分子专门调节了7TM受体受体抑制M-流动活性。这种工具的发现将促进我​​们理解7TM受体在通过M电流调节神经元兴奋性中的作用，并可能揭示KV7靶标的新型治疗机会。此外，随着对功能选择性和背景依赖性药理学的越来越多的欣赏，对新的7TM受体测定技术的需求非常需要，这些技术反映了标准测定技术目前未解决的已知生理相关效应子（例如，细胞内钙通量）。所提出的M-电流测定仅代表了这样一种新颖的测定系统。 In fact, though intracellular calcium flux is the most common cell-based functional assay technology for Gq-coupled muscarinic receptors, it appears that it is not the major signal transduction modality for muscarinic responses in some tissues including the neurons of the superior cervical ganglion (Hernandez et al, 2008) where muscarinic modulation of M-current is known to occur.因此，所提出的测定系统不仅是发现新颖和重要的小分子探针的机会，而且还代表了表征毒蕈碱受体调节剂的重要新机制。 公共卫生相关性：拟议的研究着重于新技术的开发，以便发现神经传播者受体与神经元电压门控离子通道之间相互作用的化学调节剂。这些受体和通道在神经系统功能中起关键作用，并参与引起和治疗多种神经系统疾病。拟议的研究将促进我们对这些受体和渠道在正常和病理过程中的作用的了解，重点是将它们转化为疾病疗法。