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

描述（由申请方提供）：本研究的总体目的是鉴定双孔结构域钾通道TREK-2的特异性和强效调节剂。TREK-2通道是细胞电兴奋性的重要调节剂，并发挥多种生理作用。TREK-2主要在神经系统和胰腺中表达，在DRG神经元和神经内分泌细胞中强表达。因此，TREK-2通道活性可以调节感觉神经元的刺激检测和神经内分泌激素分泌。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。因此，该项目将在高通量筛选（HTS）中利用稳健的基于铊（Tl+）的荧光测定法来鉴定人TREK-2通道的小分子调节剂。该试验将在四环素诱导型TREK-2细胞系上进行，该细胞系是根据其在Tl+试验中的性能选择的。TREK-2 T1+测定用两个小分子文库的初步筛选进行验证，所述两个小分子文库包括Spectrum Collection（~2000个分子）和生物活性脂质文库（~1000个分子），其鉴定了TREK-2的一小群分子调节剂。利用初级筛选来优化用于在大HTS中与TREK-2细胞系一起使用的T1+测定。在这些初步研究的基础上，该提案计划在分子库探针中心网络内的约翰霍普金斯离子通道中心的多样化小分子库上对人类TREK-2通道进行HTS。这将使用1。基于T1+通量的HTS，其之后将是2.利用Tl+通量以及电生理学的二次测定以支持快速命中到引线进展，并且最后3.一系列测试，包括生物物理分析、诱变和磷酸化分析以及T1+通量;这些将确定TREK-2的小分子调节剂的作用机制、特异性和效力。在本HTS中鉴定的TREK-2的分子调节剂将用于测试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.