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

描述(申请人提供)：离子通道是一种膜蛋白，它选择性地将离子传导到细胞膜上，既存在于可兴奋细胞中，也存在于不可兴奋细胞中。离子通道在细胞生理中起着关键作用，包括电和细胞信号、离子动态平衡和激素分泌。这项建议的目的是寻找小分子化学探针，激活或增强致病钾通道-KCNQ1。该通道的异常通过与不同组织中的其他附属亚单位共同组装，被认为是心脏疾病和上皮细胞疾病的原因，如长QT综合征和囊性纤维化。我们已经开发了细胞系，在基于荧光的铊替代通量分析中验证了细胞系，并进行了高通量筛选的可行性试验。这份提案概述了一个具体的计划，即使用KCNQ1细胞系进行一项包含300,000个化合物的筛选，以寻找特定的激活剂/增效剂。然后，将通过自动膜片钳记录表达不同组合的KCNQ1/KCNE通道的不同细胞系，包括KCNE1、KCNE2、KCNE3、KCNE4和KCNE5 b亚基来评估活性化合物。由于不同的KCNE亚基与KCNQ1在不同的组织中共同组装，因此根据这一建议开发的特定探针将对组织特异性研究有用。在约翰霍普金斯医学院心脏科的Gordon Tomaselli博士和霍普金斯大学上皮疾病中心的Mark Donowitz博士的合作下，我们的化合物验证计划包括在本地制剂中测试分离的化合物。随着药理和药物化学的进一步研究，这些活性化合物可能被开发用于治疗心律失常和上皮组织水盐失衡。 公共卫生相关性：非神经性KCNQ1钾通道是心跳、听力以及肺和肠道组织水/盐平衡的关键功能组件。这些钾通道的功能障碍会导致严重的心律失常、心跳骤停、听力损失、腹泻和囊性纤维化。越来越多的证据表明它们与长QT综合征、家族性房颤、Jervell和Lange-Nielsen综合征、Romano-Ward综合征、短QT综合征和婴儿猝死综合征(SIDS)之间存在因果关系。因此，通过该项目发现小分子探针可以为更好地了解这些途径提供工具，改善对未来药物开发中的安全性问题的评估，并开发上述疾病的治疗方法。

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.