Development of Bioactive Chemical Probes for Calcium-activated Chloride Channel

描述（由申请人提供）：离子通道是一种膜蛋白，存在于可兴奋细胞和不可兴奋细胞中。它们选择性地引导离子穿过细胞膜，并在细胞生理学中发挥关键作用，包括电和细胞信号，离子稳态和激素分泌。本提案的目的是为编码钙活化氯离子通道（CaCC）的TMEM16A找到有效和特异性的小分子化学探针。CaCC异常被认为是导致囊性纤维化、哮喘、慢性支气管炎和高血压等疾病的原因。它的作用涉及许多生理过程，包括心脏和神经元兴奋、感觉转导、跨上皮分泌、平滑肌收缩和受精。与首次克隆TMEM16A基因的Dr. Lily Jan的实验室合作，我们开发了几种细胞系和基于荧光的碘化物替代通量测定。高通量筛选方案正在测试CaCC靶TMEM16A。本提案概述了使用TMEM16A表达细胞系和遗传编码的Cl-/I-生物传感器进行bbb30万化合物筛选的计划，以搜索有效的CaCC抑制剂/激活剂探针。然后通过自动膜片钳记录来评估活性化合物。与加州大学旧金山分校的Lily Jan博士合作，我们未来的计划包括分别在天然制剂中测试分离的化合物，如神经元和上皮。这些活性化合物，在药理学和药物化学方面的进一步努力，可以作为工具或先导化合物用于开发治疗ccc相关疾病的药物。

DESCRIPTION (provided by applicant): Ion channels are membrane proteins, found in both excitable cells and non-excitable cells. They selectively conduct ions across cellular membranes and 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 potent and specific small molecule chemical probes for TMEM16A that encodes the calcium-activated chloride channel (CaCC). Abnormality of CaCC is thought to be causal to diseases, such as cystic fibrosis, asthma, chronic bronchitis, and hypertension. Its role is implicated in numerous physiological processes including cardiac and neuronal excitation, sensory transduction, trans-epithelial secretion, smooth muscle contraction, and fertilization. In collaboration with Dr. Lily Jan's laboratory, which first cloned the TMEM16A gene, we have developed several cell lines and a fluorescence-based iodide surrogate flux assay. The protocol for high-throughput screening is being tested for the CaCC target TMEM16A. This proposal outlines a plan to conduct a >300,000-compound screen using the TMEM16A expressing cell line with a genetically encoded Cl-/I- biosensor to search potent inhibitor/activator probes for CaCC. The active compounds will then be evaluated by automated patch-clamp recording. In collaboration with Dr. Lily Jan at University of California at San Francisco, our future plan includes testing of the isolated compounds in native preparations, such as neurons and epithelium, respectively. These active compounds, with further efforts in pharmacology and medicinal chemistry, may be exploited either as tools or as lead compounds for developing therapeutic remedies to the CaCC-associated diseases. PUBLIC HEALTH RELEVANCE: The recent discovery of the calcium-activated chloride channel (CaCC) has paved a new path to identify small molecule probes. Abnormality of CaCC activity is thought to be causal to several major diseases, such as cystic fibrosis, asthma, chronic bronchitis, and hypertension. Its role is implicated in numerous physiological processes including cardiac and neuronal excitation, sensory transduction, trans-epithelial secretion, smooth muscle contraction, and fertilization. Therefore, specific modulation compounds are useful to aid therapeutic development.