Discovery of small molecule regulators of atrial cardiomyocyte action potential duration to restore normal cardiac rhythm in atrial fibrillation

项目总结 房颤(房颤)是最常见的心律失常，全世界有3300多万人患有房颤。 在美国有100万人。房颤会导致生活质量下降、中风和全身血栓栓塞症、心力衰竭以及 死亡率上升。使用非特异性药物或程序治疗房颤及其并发症的特点是 由于结果不令人满意和高昂的成本。获得性心脏病、心脏重构、神经激素 因素、年龄和遗传特征都与房颤的存在相关。快速、不协调的心房 心房室活动是由于心肌细胞动作电位持续时间的缩短或延长 易受损害的心肌基质，导致持续性心律失常，其特征是触发或维持电路再循环 入射或早期和/或延迟后去极化。我们最近开发了一种新奇的，高级的 用于表征心肌细胞电生理特性的透过性动力学成像和分析平台 在单细胞分辨率下，可用于对功能性人体进行高通量筛查(HTS) 心房心肌细胞来源于iPS细胞产生的Id1程序化心脏祖细胞。我们的创新 在表型筛查级联中使用这种和相关的分析方法是为了发现之前 未知的、心房特有的心肌细胞电特性和节律调节器。我们的假设是这样的 这种方法最终将为未来改善心血管疾病产生类似药物的起点 治疗学。一次HTS分析已经在384孔格式中进行了全面优化，并作为对 检测准备就绪后，已筛选出400种化合物(Kolmogorov-Smirnov D-STATISTICAL&GT；0.1)。多个点击率来自 在浓度响应实验中识别并确认并验证了先导筛选。一块电池 已经开发并试行了一系列下游检测方法，以建立关键路径检测漏斗。几个 从飞行员筛选中鉴定出的化合物被测试以确定它们是否影响动作电位持续时间 KCNA5基因E375X突变致敏的心房肌细胞的变化及其对作用的影响 野生型和原代房室心肌细胞的潜伏期。这项建议建立在数据的基础上 从申请者中，来自SBP(Colas博士和Larson博士)的一个具有基础生物学和药物发现的成熟团队 该领域的专业知识和获得所有必要技术的机会。这项提议的总体目标是产生 化学生物学的研究工具和新药的起点。由于关键路径分析已全部到位，我们 预计我们可以快速获得这样的探针分子，并开始探索它们的活性。我们未来的计划是 最终确定HITS对HIT-to-Lead活动的适用性，开始在动物体内评估先导化合物 模型和最终的患者，并确定他们的细胞作用机制。这笔赠款的工作成果将是 作为提交Hit-to-Lead(HTL)授权申请和提交家长R01授权申请的初步数据 对它们的生物学机制的理解。

PROJECT SUMMARY Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia, afflicting over 33 million people worldwide and 6 million in the US. AF causes reduced quality of life, stroke and systemic thromboembolism, heart failure, and increased mortality. Treatment of AF and its complications with nonspecific drugs or procedures is characterized by unsatisfactory outcomes and significant cost. Acquired heart disease, cardiac remodeling, neurohormonal factors, aging, and genetic traits have all been correlated with presence of AF. Rapid, uncoordinated atrial chamber activity is due to shortened or prolonged cardiomyocyte action potential durations acting within a vulnerable myocardial substrate, causing persistent arrhythmia that features triggering or sustaining circuit re- entry or early and/or delayed after-depolarizations, respectively. We have recently developed a novel, high throughput kinetic imaging and analysis platform to characterize cardiomyocyte electrophysiological properties at single cell resolution, which can be used to conduct high throughput screening (HTS) on functional human atrial cardiomyocytes derived from Id1-programmed cardiac progenitors created from iPS cells. Our innovation is the use of this and related assays in a phenotypic screening cascade designed to discover previously unknown, atrial-specific modulators of cardiomyocyte electrical properties and rhythm. Our hypothesis is this approach will ultimately generate drug-like starting points for future disease-modifying cardiovascular therapeutics. The primary HTS assay has been fully optimized in a 384-well format, and as a demonstration of assay readiness, 400 compounds have been screened (Kolmogorov-Smirnov D-statistic >0.1). Multiple hits from pilot screens were identified and were confirmed and validated in concentration response experiments. A battery of downstream assays has been developed and piloted to establish a critical path-testing funnel. Several compounds identified from the pilot screen were tested to determine if they affected the action potential duration of atrial cardiomyocytes sensitized by the E375X mutation in KCNA5, and if they had effects on the action potential durations of wild type and primary atrial and ventricular cardiomyocytes. This proposal builds on data from the applicants, an established team from SBP (Drs. Colas and Larson) with basic biology and drug discovery expertise in the field and access to all necessary technologies. The overall goal of this proposal is to generate chemical biology research tools and starting points for new drugs. As the critical path assays are all in place, we anticipate we can rapidly obtain such probe molecules and start to explore their activity. Our future plans are to ultimately determine hits’ suitability for hit-to-lead activities, begin in vivo evaluation of lead compounds in animal models and eventually patients, and determine their cellular mechanism of action. This grant’s work product will serve as preliminary data for hit-to-lead (HTL) grant submissions and parent R01 grant submissions to pursue understanding of their biological mechanisms.