Small molecule modulators of the two-pore-domain potassium channel, TREK-2

描述（由申请人提供）：本研究的总体目标是确定两孔域钾通道TREK-2的特异性和有效调节剂。TREK-2通道是细胞电兴奋性的重要调节因子，具有多种生理功能。TREK-2主要表达于神经系统和胰腺，在DRG神经元和神经内分泌细胞中表达较强。因此，TREK-2通道的活性可能调节感觉神经元的刺激检测和神经内分泌激素的分泌。然而，由于缺乏特异性和有效的药理学，我们对TREK-2在人体组织中的作用的理解仍然模糊不清。因此，本项目将在高通量筛选（HTS）中利用基于铊（Tl+）的荧光分析来鉴定人类TREK-2通道的小分子调节剂。该试验将在四环素诱导的TREK-2细胞系上进行，该细胞系是根据其在Tl+试验中的表现而选择的。TREK-2 Tl+试验通过两个小分子文库的初级筛选进行了验证，包括光谱收集（~2000个分子）和生物活性脂质文库（~1000个分子），确定了TREK-2的一小群分子调节因子。利用初级筛选优化TREK-2细胞系在大HTS中的Tl+测定。在这些初步研究的基础上，本提案计划利用分子文库探针中心网络中约翰霍普金斯离子通道中心的各种小分子文库对人类TREK-2通道进行HTS。这将使用1来完成。一个基于Tl+通量的高温超导，接下来是2个。二次分析利用Tl+通量和电生理学，以支持快速的命中导联进展，最后3。一系列测试，包括生物物理分析、诱变和磷酸化分析以及Tl+通量；这些将决定TREK-2小分子调节因子的作用机制、特异性和效力。本次HTS鉴定的TREK-2分子调控因子将用于检测TREK-2通道对人胰岛细胞电活动和激素分泌的影响。

DESCRIPTION (provided by applicant): The overall objective of this study is to identify specific and potent modulators of the two-pore-domain potassium channel, TREK-2. TREK-2 channels are important regulators of cellular electrical excitability and serve diverse physiological roles. TREK-2 is expressed predominately in the nervous system and pancreas, with strong expression in DRG neurons and neuroendocrine cells. TREK-2 channel activity may, thus, regulate stimuli detection of sensory neurons and neuroendocrine hormone secretion. However, our understanding of the role(s) of TREK-2 in human tissues remains obscure due to a lack of specific and potent pharmacology. Therefore, this project will utilize a robust thallium (Tl+) based fluorescent assay in a high throughput screen (HTS) to identify small molecule modulators of the human TREK-2 channel. The assay will be performed on a tetracycline inducible TREK-2 cell line, which was selected for based on its performance in the Tl+ assay. The TREK-2 Tl+ assay was validated with primary screens of two small molecule libraries including the Spectrum Collection (~2000 molecules) and a bioactive lipid library (~1000 molecules), which identified a small cohort of molecular regulators of TREK-2. The primary screens were utilized to optimize the Tl+ assay for use with the TREK-2 cell line in a large HTS. Building on these preliminary studies, this proposal plans to perform a HTS on the human TREK-2 channel with the diverse small molecule library at the Johns Hopkins Ion Channel Center within the Molecular Libraries Probe Centers Network. This will be accomplished using 1. A Tl+ flux based HTS, which will be followed by 2. Secondary assays utilizing Tl+ flux as well as electrophysiology to support rapid hit-to-lead progression and finally 3. A battery of tests including biophysical analysis, mutagenesis and phosphorylation analysis together with Tl+ flux; these will determine the mechanism of action, specificity and potency of the small molecule regulators of TREK-2. Molecular regulators of TREK-2 identified in this HTS will be utilized to test the influence of TREK-2 channels on human islet cell electrical activity and hormone secretion.