Development of Chemical Probes for Two Pore Potassium Channels

描述(申请人提供)：两个孔结构域钾通道(K2p‘s)分为六个亚家族：Twik、Trek、TASK、Talk、Thik和Tresk。它们是由具有两个串联孔结构域的亚基二聚形成的，每个结构域类似于电压门控钾通道的结构域。TASK3是任务子家族的一员，在早期对组织分布、电生理学和非特异性药物干预的研究基础上，提出了感知代谢水平、赋予麻醉敏感性、调节神经元兴奋性和癌细胞增殖的功能。因此，TASK3被认为是治疗癌症和神经系统疾病的潜在靶点。然而，由于缺乏选择性配体，对该通道的研究进展非常缓慢。我们建议对专门针对TASK3通道的化合物进行高通量筛选，并进一步将它们开发成小分子探针。在我们的初步研究中，我们构建了一个表达TASK3的HEK-293细胞系，并在FDSS上优化了基于Tl+的384孔式荧光分析。此外，我们还利用野生型HEK-293细胞株和表达Thik-2和其他钾通道的HEK-293细胞株开发了电生理二级筛选和反筛选。使用Sigma-Aldridge的Lopac文库进行的验证筛选导致了一种化合物N1786的鉴定，该化合物抑制TASK3。与TASK3特异相互作用的小分子探针可能成为研究这些独特离子通道的结构和功能以及开发靶向治疗方法的有价值的工具。 与公共健康相关：两孔结构域钾通道(K2p‘s)在调节兴奋性、感知环境条件以及调节细胞分泌和增殖方面发挥着重要作用。特别是，TASK3被认为是癌症和神经疾病的潜在治疗靶点。然而，由于缺乏特定的配体，人们对这些通道知之甚少。我们建议对专门针对TASK3通道的化合物进行高通量筛选，并进一步将它们开发成小分子探针。

DESCRIPTION (provided by applicant): The two pore domain potassium channels (K2p's) are divided into six subfamilies: TWIK, TREK, TASK, TALK, THIK, and TRESK. They are formed by dimerization of subunits that have two tandem pore domains each resembling that of the voltage-gated potassium channels. TASK3 is a member of the TASK subfamily, and has proposed functions of sensing metabolic level, conferring anesthetic sensitivity, as well as modulating neuronal excitability and cancer cell proliferation, based on early studies on tissue distribution, electrophysiology, and manipulative intervention with non-specific reagents. Therefore, TASK3 has been considered a potential therapeutic target for cancer and neurological disorders. However, due to the lack of selective ligands, progress in the study on this channel has been very slow. We propose to perform a high-throughput screen for compounds that specifically target TASK3 channels, and further develop them into small molecular probes. In our preliminary studies, we have constructed a HEK-293 cell line expressing TASK3, and optimized a Tl+ - based fluorescence assay in 384-well format on FDSS. In addition, we have also developed secondary screens with electrophysiology and counter screens using wild-type HEK-293 cell line, and HEK-293 expressing THIK-2 and other potassium channels. A validation screen using the LOPAC library from Sigma- Aldridge has led to the identification of a compound, N1786, that inhibits TASK3. Small molecular probes that specifically interact with TASK3 may become valuable tools for studying the structure and functions of these unique ion channels, and developing targeted therapeutic approaches. PUBLIC HEALTH RELEVANCE: The two-pore-domain potassium channels (K2p's) play important roles in modulating excitability, sensing environmental conditions, as well as regulating cellular secretion and proliferation. In particular, TASK3 has been considered a potential therapeutic target for cancer and neurological disorders. However, very little is known about these channels, because of the lack of specific ligands. We propose to perform a high-throughput screen for compounds that specifically target TASK3 channels, and further develop them into small molecular probes.