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

描述(由申请人提供)：这项研究的总体目标是确定两孔结构域钾通道Trek-2的特定和有效的调节剂。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有较强的表达。因此，Trek-2通道活动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，由于缺乏特定和有效的药理作用，我们对Trek-2在人体组织中的作用(S)的了解仍然不清楚。因此，该项目将在高通量筛选(HTS)中利用基于Tl+的强大的荧光分析来识别人类Trek-2通道的小分子调节器。该检测将在四环素可诱导的Trek-2细胞系上进行，该细胞系是根据其在TL+检测中的表现而被选中的。对两个小分子文库(光谱文库(~2000个分子)和生物活性脂类文库(~1000个分子))的初步筛选验证了Trek-2 TL+实验的有效性，该文库鉴定了一小部分Trek-2的分子调控因子。利用初步筛选来优化TL+试验，以用于大型HTS中的Trek-2细胞系。在这些初步研究的基础上，这项提案计划在分子图书馆探测中心网络内的约翰霍普金斯离子通道中心利用不同的小分子文库对人类Trek-2通道进行高温超导。这将使用1.基于TL+焊剂的HTS，随后将是2.利用TL+焊剂以及电生理学来支持快速Hit-to-Lead进展的二次检测，最后是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+焊剂；这些将确定Trek-2小分子调节剂的作用机制、特异性和效力。在本HTS中确定的Trek-2的分子调节器将被用于测试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.