High Throughput Screening for Chemical Modifiers of Long QT Syndrome

描述（由申请人提供）：先天性长QT综合征（LQTS）是一种遗传性疾病，心律失常导致心源性猝死的风险增加。LQTS每3000人中就有1人受到影响，每年导致约4000人死亡。虽然我们对LQTS的分子发病机制的理解取得了重大进展，但治疗选择仍然有限。主要的药物治疗仍然是β -阻滞剂，它通常对危及生命的心律失常提供不完全和不可靠的保护。因此，许多患者需要植入式心律转复除颤器，这是有创的，并且具有显着的短期和长期风险，包括不适当的电击和感染。由于LQT患者在发病时往往很年轻，他们承担了多次ICD发生器改变和导联修订的风险和发病率。心脏复极在物种间的显著差异为LQTS动物模型的鉴定带来了重大障碍。在过去的几年里，我们的实验室为斑马鱼心脏电生理模型的发展做出了贡献。利用这个模型，我们已经证明了复极的遗传和化学扰动的忠实再现。斑马鱼突变体霹雳舞携带钾通道基因KCNH2突变，该基因在人类长QT2型（LQT2）中发生突变。2型LQTS约占所有人类长QT病例的三分之一。KCNH2编码通道也是迄今为止发现的每一种QT延长药物的靶点。在初步数据中，我们表明斑马鱼霹雳舞突变体再现了人类长QT2综合征的几个关键特征。这种霹雳舞突变I59S导致一种蛋白质不经历复杂的糖基化或转运到质膜，这与迄今为止研究的大多数人类LQT2病例相似。在1200个小分子的人工先导筛选中，我们已经确定了两种抑制斑马鱼LQT2表型的化合物。在二次筛选中，我们证明了这些化合物在LQT2模型中缩短了心室动作电位持续时间。这些来自小型人工试验筛检的初步结果支持大规模筛检工作。在本提案中，我们详细计划自动化我们的斑马鱼LQT测定，以确定抑制斑马鱼长QT表型的小分子，具体目的如下：开发一种检测斑马鱼LQT表型的自动检测方法。2. 利用斑马鱼LQT模型验证全生物HTS可行的假设。由于心脏电生理的生物学复杂性以及长QT综合征缺乏有效的药物靶点，需要在完整的机体中进行筛查。本应用程序中描述的检测将是发现小分子和治疗靶点的独特工具，专门针对LQTS中潜在的生理缺陷，服务于尚未满足的临床需求。

DESCRIPTION (provided by applicant): Congenital long QT syndrome (LQTS) is a heritable disease that carries an increased risk of sudden cardiac death due to cardiac arrhythmia. LQTS affects 1 in 3000 individuals and results in approximately 4,000 deaths annually. While significant advances have been made in our understanding of the molecular pathogenesis of LQTS, treatment options remain limited. The mainstay of pharmacologic therapy remains beta-blockade, which often provides incomplete and unreliable protection against life-threatening arrhythmias. Thus, many patients require implantable cardioverter defibrillators which are invasive, and carry significant short and long-term risks including inappropriate shocks and infection. Because LQT patients are often young at presentation they bear the risk and morbidity of multiple ICD generator changes and lead revisions. Marked interspecies variation in cardiac repolarization has created significant barriers in identification of animal models of LQTS. Over the last several years, our laboratory has contributed to the development of a zebrafish model of cardiac electrophysiology. Using this model we have demonstrated the faithful reproduction of both genetic and chemical perturbations of repolarization. The zebrafish mutant break-dance carries a mutation in the potassium channel gene, KCNH2, which is the gene mutated in human long QT type 2 (LQT2). Type 2 LQTS is responsible for approximately one third of all human long QT cases. The KCNH2 encoded channel is also the target of every QT prolonging drug identified to date. In preliminary data we show that the zebrafish break-dance mutant recapitulates several key features of human long QT2 syndrome. The break-dance mutation, I59S, results in a protein that does not undergo complex glycosylation or trafficking to the plasma membrane, similar to the majority of human LQT2 cases studied to date. In a manual pilot screen of 1200 small molecules, we have identified two compounds that suppress the zebrafish LQT2 phenotype. In a secondary screen, we demonstrate that these compounds shorten the ventricular action potential duration in our LQT2 model. These preliminary results from a small manual pilot screen support a large scale screening effort. In this proposal we detail plans to automate our zebrafish LQT assay in order to identify small molecules that suppress the zebrafish long QT phenotype in the following Specific Aims: 1. To develop an automated assay for detection of the zebrafish LQT phenotype. 2. To test the hypothesis that whole-organism HTS is feasible using the zebrafish LQT model. The biologic complexity of cardiac electrophysiology as well as the lack of validated drug targets in Long QT Syndrome demands screening in an intact organism. The assays described in this application will be unique tools for discovering small molecules and therapeutic targets that specifically address the underlying physiologic defect in LQTS, serving an as yet unmet clinical need. PUBLIC HEALTH RELEVANCE: Congenital long QT syndrome is a genetic disease that carries an increased risk of sudden cardiac death due to cardiac arrhythmia and for which current therapies are inadequate. In this proposal we detail plans to automate a zebrafish assay of long QT syndrome in order to identify new compounds that could be used to treat this disease. The studies described in this application will provide unique tools for discovering small molecules and therapeutic targets that specifically address the underlying problem in LQTS, serving an as yet unmet clinical need.