High Throughput Screening for Chemical Modifiers of Long QT Syndrome

高通量筛选长 QT 综合征的化学修饰剂

基本信息

批准号：
8154017
负责人：
David J Milan
金额：
$ 62.35万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-05 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8154017
关键词：
Action Potentials Address Affect Animal Model Arrhythmia Automation Biological Assay Cardiac Case Study Cell membrane Cells Cessation of life Chemical Modifier Chemicals Clinical Complement Complex Dancing Data Defect Detection Development Disease Drug Delivery Systems Electrocardiogram Electrophysiology (science)Environment Genes Genetic Heart Rate Hereditary Disease Human Implantable Defibrillators In Vitro Individual Infection Laboratories Lead Life Long QT Syndrome Manuals Modeling Molecular Molecular Target Morbidity - disease rate Mutate Mutation Organism Pathogenesis Patients Pharmaceutical Preparations Phenotype Physiological Potassium Channel Process Property Proteins Regulation Reproduction Risk Screening procedure Shock Syndrome Testing Therapeutic Toxic effect Ursidae Family Variant Ventricular Whole Organism Zebrafish assay development base chemical genetics glycosylation high throughput screening mutant response small molecule sudden cardiac death therapeutic target tool trafficking

项目摘要

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.

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，会增加因心律失常导致心源性猝死的风险。 LQTS 影响每 3000 人中就有 1 人，每年导致约 4,000 人死亡。虽然我们对 LQTS 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。药物治疗的支柱仍然是β-受体阻滞剂，它通常对危及生命的心律失常提供不完整且不可靠的保护。因此，许多患者需要植入式心脏复律除颤器，这是侵入性的，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 患者就诊时通常很年轻，因此他们承受着多次 ICD 发生器更换和导联修改的风险和发病率。心脏复极的显着种间差异给 LQTS 动物模型的鉴定造成了重大障碍。在过去的几年中，我们的实验室为斑马鱼心脏电生理学模型的开发做出了贡献。使用这个模型，我们证明了复极化的遗传和化学扰动的忠实再现。斑马鱼突变霹雳舞携带钾通道基因KCNH2的突变，该基因是人类长QT 2型（LQT2）的突变基因。 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确定的每种 QT 延长药物的靶标。在初步数据中，我们表明斑马鱼霹雳舞突变体概括了人类长 QT2 综合征的几个关键特征。霹雳舞突变 I59S 产生的蛋白质不会经历复杂的糖基化或运输到质膜，类似于迄今为止研究的大多数人类 LQT2 病例。在对 1200 个小分子进行的手动试点筛选中，我们鉴定出了两种抑制斑马鱼 LQT2 表型的化合物。在二次筛选中，我们证明这些化合物可以缩短 LQT2 模型中的心室动作电位持续时间。这些来自小型手动试点筛选的初步结果支持大规模筛选工作。在本提案中，我们详细介绍了斑马鱼 LQT 测定自动化的计划，以便识别抑制斑马鱼长 QT 表型的小分子，具体目标如下： 1. 开发一种用于检测斑马鱼 LQT 表型的自动化测定。 2. 使用斑马鱼 LQT 模型检验整个生物体 HTS 可行的假设。心脏电生理学的生物学复杂性以及长 QT 综合征缺乏经过验证的药物靶点需要在完整的生物体中进行筛查。本申请中描述的测定将成为发现小分子和治疗靶点的独特工具，专门解决 LQTS 的潜在生理缺陷，满足尚未满足的临床需求。公共卫生相关性：先天性长 QT 综合征是一种遗传性疾病，会增加因心律失常导致心源性猝死的风险，而目前的治疗方法对此还不够。在本提案中，我们详细介绍了对长 QT 综合征进行斑马鱼自动化检测的计划，以便识别可用于治疗这种疾病的新化合物。本申请中描述的研究将为发现小分子和治疗靶点提供独特的工具，专门解决 LQTS 的根本问题，满足尚未满足的临床需求。