Subtype-Specific Small Molecule Chemical Probes for Non-neuronal KCNQ1 Potassium

描述（由申请人提供）：离子通道是一种膜蛋白，可选择性地将离子传导到细胞膜上，存在于可兴奋细胞和不可兴奋细胞中。离子通道在细胞生理学中起着至关重要的作用，包括电和细胞信号，离子稳态和激素分泌。本提案的目的是寻找激活或增强致病钾通道KCNQ1的小分子化学探针。该通道的异常，通过与其他附属亚基在不同组织中的共组装，被认为是心脏疾病和上皮细胞疾病（如长QT综合征和囊性纤维化）的原因。我们已经开发了细胞系，在基于荧光的铊替代通量试验中验证了这些细胞系，并进行了高通量筛选的可行性试验。本提案概述了使用KCNQ1细胞系进行bbb30万化合物筛选以搜索特定激活剂/增强剂的具体计划。然后通过自动膜片钳记录各种表达KCNQ1/KCNE通道不同组合的细胞系，包括KCNE1、KCNE2、KCNE3、KCNE4和KCNE5 b-亚基，来评估活性化合物。由于不同的kcnne亚基在不同的组织中与KCNQ1共同组装，因此根据该提案开发的特定探针将有助于组织特异性研究。我们与约翰霍普金斯医学院心脏病学部门的Gordon Tomaselli博士和霍普金斯上皮疾病中心的Mark Donowitz博士合作，我们的化合物验证计划包括对天然制剂中分离化合物的测试。这些活性化合物，在药理学和药物化学方面的进一步研究，有望用于心律失常和上皮组织水盐失衡的治疗。

DESCRIPTION (provided by applicant): Ion channels are membrane proteins that selectively conduct ions across cellular membranes, existing in both excitable cells and non-excitable cells. Ion channels play a critical role in cellular physiology, including electrical and cellular signaling, ion homeostasis, and hormone secretion. The objective of this proposal is to find small molecule chemical probes that activate or potentiate a disease-causing potassium channel - KCNQ1. Abnormality of this channel, through co-assembly with other accessory subunits in different tissues, is thought to be causal to both cardiac diseases and epithelial cell diseases, such as long QT syndrome and cystic fibrosis. We have developed cell lines, validated the cell lines in a fluorescence-based thallium surrogate flux assay, and perform feasibility trials of high throughput screening. This proposal outlines a specific plan to conduct a >300,000-compound screen using the KCNQ1 cell line to search for specific activators/potentiators. The active compounds will then be evaluated by automated patch-clamp recording of various cell lines expressing different combinations of KCNQ1/KCNE channels, including KCNE1, KCNE2, KCNE3, KCNE4 and KCNE5 b- subunits. Because different KCNE subunits co-assemble with KCNQ1 in different tissues, specific probes developed from this proposal will therefore be useful for tissue-specific investigations. In collaboration with Dr. Gordon Tomaselli in Division of Cardiology and Dr. Mark Donowitz in Hopkins Center for Epithelial Disorders of Johns Hopkins School of Medicine, our compound validation plan includes testing of the isolated compounds in native preparations. These active compounds, with further efforts in pharmacology and medicinal chemistry, may be exploited for therapeutic remedy of cardiac arrhythmia and water and salt imbalance in epithelial tissues. PUBLIC HEALTH RELEVANCE: Non-neuronal KCNQ1 potassium channels are critical functional components of heartbeat, hearing, and water/salt balance in lung and intestinal tissues. Dysfunction of these potassium channels results in serious arrhythmia, cardiac arrest, hearing loss, diarrhea, and cystic fibrosis. There is increasing evidence for their causality for long QT syndrome, familial atrial fibrillation, Jervell and Lange-Nielsen syndrome, Romano-Ward syndrome, short QT syndrome, and sudden infant death syndrome (SIDS). Discovery of small molecule probes through this project can therefore provide tools for better understanding of these channels, improve assessment of safety concerns in future pharmaceutical development, and develop therapeutics for the above mentioned diseases.