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

项目概要 心房颤动 (AF) 是最常见的心律失常，全世界有超过 3300 万人受其困扰，6 万在美国。房颤会导致生活质量下降、中风和全身性血栓栓塞、心力衰竭和 死亡率增加。使用非特异性药物或手术治疗 AF 及其并发症的特点是 由于结果不令人满意和成本高昂。获得性心脏病、心脏重塑、神经激素 因素、衰老和遗传特征都与房颤的存在相关。快速、不协调的心房 心室活动是由于心肌细胞动作电位持续时间的缩短或延长所致 脆弱的心肌基质，导致持续性心律失常，其特征是触发或维持电路重新 分别为进入或早期和/或延迟后除极。我们最近开发了一种新颖的、高 用于表征心肌细胞电生理特性的通量动力学成像和分析平台 单细胞分辨率，可用于对功能性人类进行高通量筛选（HTS） 心房心肌细胞源自 iPS 细胞产生的 Id1 编程心脏祖细胞。我们的创新 是在表型筛选级联中使用该方法和相关分析，旨在发现以前的情况 心肌细胞电特性和节律的未知心房特异性调节剂。我们的假设是这样的 该方法最终将为未来改善心血管疾病产生类似药物的起点 疗法。主要 HTS 测定已在 384 孔格式中进行了全面优化，并作为演示 分析准备就绪，已筛选出 400 种化合物（Kolmogorov-Smirnov D 统计量 >0.1）。多次点击来自 确定了试点筛选，并在浓度响应实验中进行了确认和验证。一块电池 下游分析的开发和试点已建立一个关键的路径测试漏斗。几个 对中试筛选中鉴定出的化合物进行了测试，以确定它们是否影响动作电位持续时间 心房心肌细胞对 KCNA5 中 E375X 突变敏感的情况，以及它们是否对作用有影响 野生型和原代心房和心室心肌细胞的潜在持续时间。该提案建立在数据的基础上 来自申请人的 SBP（Colas 博士和 Larson 博士）的一支既定团队，具有基础生物学和药物发现能力 该领域的专业知识和获得所有必要技术的机会。该提案的总体目标是产生 化学生物学研究工具和新药的起点。由于关键路径分析均已到位，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 最终确定命中对先导化合物活性的适用性，开始对动物先导化合物进行体内评估 模型和最终患者，并确定其细胞作用机制。这笔赠款的工作产品将 作为点击先导 (HTL) 拨款申请和父 R01 拨款申请的初步数据 了解它们的生物学机制。

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.