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

描述（由申请人提供）：m电流大约在30年前首次被描述（Brown and Adams, 1980），现在被认为是许多神经过程的关键调节器，在控制兴奋性方面起着主导作用。近年来，已经报道了一些M-电流（电压门控离子通道的Kv7家族）下的蛋白质的小分子调节剂。该通道的激活剂和抑制剂已经被描述过，至少有一种激活剂，雷沙滨，已经进入晚期临床试验，用于治疗癫痫。多年来，m电流的机制（Gq偶联毒蕈碱乙酰胆碱受体的激动剂抑制电流）一直是个谜。然而，Suh和Hille（2002）的研究表明，毒蕈碱受体刺激质膜PIP2耗竭可能是毒蕈碱受体抑制m电流的机制。在随后的几年里，这种调制m电流的机制一直是研究的热点。然而，迄今为止还没有关于易于使用的报告；hts兼容的分析，以评估m电流调制。也没有任何小分子工具报道专门针对7TM受体调节这种至关重要的电导的能力。为了解决这个问题，我们已经开始努力开发一个hts兼容的分析系统和一套二级分析，以使筛选专注于发现和表征特异性调节7TM受体抑制m电流活性的小分子。这些工具的发现将促进我们对7TM受体在通过m电流调节神经元兴奋性中的作用的理解，并可能为Kv7靶点提供新的治疗机会。此外，随着功能选择性和环境依赖性药理学的不断发展，迫切需要新的7TM受体检测技术，以反映目前标准检测技术无法解决的已知生理相关效应（例如细胞内钙通量）。所提出的m电流测定法就是这样一种新颖的测定系统。事实上，虽然细胞内钙通量是gq偶联毒蕈碱受体最常见的基于细胞的功能检测技术，但在包括颈上神经节神经元在内的一些组织中，它似乎不是毒蕈碱反应的主要信号转导方式（Hernandez et al, 2008），在这些组织中，已知会发生毒蕈碱对m电流的调制。因此，所提出的检测系统不仅代表了发现新颖和重要的小分子探针的机会，它也代表了表征毒蕈碱受体调节剂的重要新机制。

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.