TRPM2 HTS Screening Assay Development(RMI)

描述（由申请人提供）：TRPM2 Ca2+渗透性阳离子通道在adp核糖与明确定义的c端门控域结合时打开。TRPM2已被证明参与氧化应激诱导的钙进入，其中持续的TRPM2通道打开是由于线粒体作为第二信使释放adp核糖（47）。关于trpm2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有趣，因为TRPM2在纹状体神经元和胰腺β细胞中都被发现，这些细胞类型已知特别容易受到氧化应激的影响或损伤。然而，目前还没有TRPM2缺乏症的动物模型，也没有能够特异性调节氧化应激诱导的TRPM2介导的钙信号的小分子。氧化应激诱导的TRPM2钙信号的特异性小分子调节剂的发展将为了解TRPM2在氧化应激的生理和病理生理中的作用提供重要的工具，并有可能有助于开发临床有用的治疗方法，用于氧化应激诱导的TRPM2门控涉及细胞损伤的病理生理情况。在我们对TRPM2功能的研究过程中，我的实验室开发了一种HEK-293细胞系（HEKTRPM2细胞），它组成性地表达了高水平的人类TRPM2通道，其生物物理特性与天然的人类TRPM2没有区别（45）。HEK-TRPM2细胞系生长迅速，在许多传代中表现出稳定的TRPM2表达，并且具有粘附性，其特性适合用于荧光报告初级HTS筛选和中等通量膜片钳试验，用于二次筛选，命中优化和特异性筛选。在这项R21授权中，我们建议使用HEK-TRPM2细胞系开发优化的检测方法，用于高通量小分子筛选trpm2介导的钙信号调节剂。在Specific Aim 1中，我们将比较HEK-TRPM2细胞对荧光钙指示剂和膜电位报告器的反应，以确定哪种读数最适合TRPM2调节剂的HTS筛选。在Specific Aim 2中，我们将利用在Specific Aim 1中鉴定的荧光报告系统，并将其用于96和384孔板格式的HEK-TRPM2细胞。使用96孔板格式，我们还将使用已知生物活性化合物库评估整体方法的性质和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.