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

描述（由申请人提供）：M电流在大约30年前首次被描述（Brown和亚当斯，1980），现在被认为是许多神经过程的关键调节因子，在控制兴奋性方面起主导作用。近年来，已经报道了作为M-电流基础的蛋白质（电压门控离子通道的Kv 7家族）的许多小分子调节剂。已经描述了通道的激活剂和抑制剂，并且至少一种激活剂Retigabine已经进展到用于治疗癫痫的后期临床试验。多年来，M-电流名称背后的机制（通过Gq偶联的毒蕈碱乙酰胆碱受体激动剂抑制电流）仍然是个谜。然而，Suh和Hille（2002）的工作揭示了毒蕈碱受体刺激的质膜PIP 2的消耗是控制M-电流的毒蕈碱受体抑制的可能机制。在随后的几年中，这种调制M电流的机制一直受到强烈的研究兴趣。然而，到目前为止，还没有易于使用的报告; HTS-兼容的测定来评估M-电流调制。也没有报道任何小分子工具特异性靶向7 TM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兼容的测定系统和一套辅助测定，以使筛选集中在发现和表征的小分子，特异性调节7 TM受体抑制M-电流活性。这些工具的发现将促进我们对7 TM受体通过M电流调节神经元兴奋性的作用的理解，并可能揭示Kv 7靶点的新治疗机会。此外，随着对功能选择性和环境依赖性药理学的日益认识，强烈需要新的7 TM受体测定技术，其反映目前未通过标准测定技术解决的已知生理学相关效应物（例如细胞内钙通量）。所提出的M-电流测定代表了这样一种新颖的测定系统。事实上，尽管细胞内钙通量是Gq偶联毒蕈碱受体的最常见的基于细胞的功能测定技术，但似乎它不是某些组织中毒蕈碱应答的主要信号转导模式，包括已知M电流发生毒蕈碱调节的上级颈神经节的神经元（埃尔南德斯等人，2008）。因此，所提出的测定系统不仅代表了发现新的和重要的小分子探针的机会，它还代表了表征毒蕈碱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剂。这些受体和通道在神经系统功能中起关键作用，并涉及引起和治疗许多神经系统疾病。拟议的研究将推进我们对这些受体和通道在正常和病理过程中的作用的认识，重点是将其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.