TRPM2 HTS Screening Assay Development(RMI)

描述（由申请方提供）：TRPM 2 Ca 2+渗透性阳离子通道响应ADP-核糖与明确定义的C-末端门控结构域的结合而打开。TRPM 2已被证明参与氧化应激诱导的钙内流，其中持续的TRPM 2通道开放是由于线粒体释放ADP-核糖作为第二信使（47）。关于TRPM 2依赖性钙信号的一个主要问题是它们在氧化应激诱导的细胞反应中所起作用的性质。在原代细胞类型和体内动物模型中解决这类问题特别令人感兴趣，因为TRPM 2已在纹状体神经元和胰腺β细胞中发现，已知这些细胞类型特别容易受到氧化应激的影响或损伤。然而，目前还没有TRPM 2缺乏的动物模型，并且目前还没有能够特异性调节由TRPM 2介导的氧化应激诱导的钙信号的小分子。开发氧化应激诱导的TRPM 2钙信号的特异性小分子调节剂将为理解TRPM 2在氧化应激的生理学和病理生理学中的作用提供重要的工具，并有可能有助于开发临床上有用的治疗方法，用于氧化应激诱导的TRPM 2门控与细胞损伤有关的病理生理学情况。在我们研究TRPM 2功能的过程中，我的实验室开发了一种HEK-293细胞系（HEKTRPM 2细胞），其组成型表达高水平的人TRPM 2通道，其生物物理特性与天然人TRPM 2的生物物理特性无法区分（45）。HEK-TRPM 2细胞系生长迅速，在多次传代中表现出稳定的TRPM 2表达，并且具有粘附性，其性质与其在荧光报告基因初级HTS筛选和中等通量膜片钳测定中的使用相容，所述测定是二级筛选、命中优化和特异性筛选所需的。在这项R21授权中，我们建议使用HEK-TRPM 2细胞系开发优化的检测方法，用于高通量小分子筛选TRPM 2介导的钙信号调节剂。在具体目标1中，我们将比较HEK-TRPM 2细胞对荧光钙指示剂和膜电位报告物的反应，以确定哪种读数最适合HTS筛选TRPM 2调节剂。在Specific Aim 2中，我们将利用Specific Aim 1中鉴定的荧光报告系统，并将其与HEK-TRPM 2细胞一起用于96孔和384孔板形式测定。使用96孔板格式，我们还将使用已知生物活性的化合物库评价总体方法的特性和对HTS的适用性。

DESCRIPTION (provided by applicant): The TRPM2 Ca2+-permeable cation channel opens in response to binding of ADP-ribose to a well defined C-terminal gating domain. TRPM2 has been shown to be involved in oxidative-stress induced calcium entry, where sustained TRPM2 channel opening results from release of ADP-ribose by mitochondria as a 2nd messenger (47). A major question regarding TRPM2-dependent calcium signals is the nature of the role they play in oxidative-stress induced cellular responses. Addressing this type of question in primary cell types and in vivo animal models is of particular interest, as TRPM2 has been found in both striatal neurons and pancreatic beta cells, cell types which are known to be especially susceptible to influence or damage by oxidative stress. However, no animal models of TRPM2-deficiency are presently available, and no small molecules are presently available which are capable of specifically modulating oxidative-stress induced calcium signals mediated by TRPM2. The development of specific small molecule modulators of oxidative stress induced TRPM2 calcium signals would provide important tools for use in understanding TRPM2's role in the physiology and pathophysiology of oxidative stress, and has the potential to contribute to the development of clinically useful therapeutics for pathophysiologic situations in which oxidative stress-induced TRPM2 gating is implicated in cell damage. In the course of our work on TRPM2 function, my laboratory has developed an HEK-293 cell line (HEKTRPM2 cells) which constitutively expresses a high level of human TRPM2 channels, the biophysical properties of which are indistinguishable from those of native human TRPM2 (45). The HEK-TRPM2 cell line grows rapidly, has exhibited stable TRPM2 expression over many passages, and is adherent, properties compatible with its use in both fluorescent reporter primary HTS screens and medium-throughput patch clamp assays required for secondary screening, hit optimization and specificity screening. In this R21 grant, we propose to use the HEK-TRPM2 cell line to develop optimized assays for use in high throughput small molecule screens for modulators of TRPM2-mediated calcium signals. In Specific Aim 1, we will compare HEK-TRPM2 cell responses to fluorescent calcium indicators and membrane potential reporters to determine which readout is best suited to HTS screening for TRPM2 modulators. In Specific Aim 2, we will utilize the fluorescent reporter system identified in Specific Aim 1 and adapt its use with HEK-TRPM2 cells for 96 and 384 well plate format assays. Using the 96 well plate format, we will also evaluate the overall method's properties and suitability for HTS using a library of compounds of known bioactivity.