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

描述(申请人提供)：M-电流在大约30年前首次被描述(Brown和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。最近几年，已经报道了一些M电流(电压门控离子通道的KV7家族)下面的蛋白质的小分子调节剂。该通道的激活剂和抑制剂都已被描述，至少有一种激动剂，雷替加宾，已经进入治疗癫痫的晚期临床试验。多年来，M-电流命名的机制(GQ偶联的M-乙酰胆碱受体激动剂对该电流的抑制)一直是个谜。然而，Suh和Hille(2002)的工作揭示了M受体刺激的质膜PIP2的耗竭可能是M受体抑制M-电流的机制。在接下来的几年里，这种调制M电流的机制引起了人们的强烈研究兴趣。然而，到目前为止，还没有关于易于使用的、与HTS兼容的测试方法来评估M-电流调制的报道。也没有任何小分子工具专门针对7TM受体调节这一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种与HTS兼容的检测系统和一套二次检测，以实现专注于发现和表征特定调节7TM受体抑制M-电流活性的小分子的屏幕。这类工具的发现将促进我们对7TM受体通过M-电流调节神经元兴奋性的作用的理解，并可能为KV7靶点提供新的治疗机会。此外，随着对功能选择性和上下文依赖药理学的日益认识，迫切需要新的7TM受体检测技术来反映标准检测技术(例如细胞内钙通量)目前尚未涉及的已知生理相关效应。所提出的M-电流检测就是这样一种新的检测系统。事实上，尽管细胞内钙通量是GQ偶联的M受体最常见的基于细胞的功能分析技术，但它似乎不是某些组织中M电流的主要信号转导方式，包括颈上神经节的神经元(Hernandez等，2008)，在那里M电流发生M-电流的M-电流调制。因此，所提出的检测系统不仅代表了发现新的和重要的小分子探针的机会，而且也代表了表征M受体调节剂的重要的新机制。 公共卫生相关性：拟议的研究重点是开发新技术，以发现神经递质受体和神经元电压门控离子通道之间相互作用的化学调节器。这些受体和通道在神经系统功能中起着关键作用，并与导致和治疗许多神经系统疾病有关。这项拟议的研究将促进我们对这些受体和通道在正常和病理过程中的作用的了解，重点是将这些转化为疾病的治疗方法。

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.