Discovery of small molecule promoters of cardiomyocyte proliferation to restore cardiac performance in disease

标题：发现心肌细胞增殖小分子促进剂以修复心脏 疾病中的表现 项目总结 心血管疾病是发达国家和发展中国家的主要死亡原因。现有的CVD 药物治疗方案不能治疗心肌及其成分中的任何基础问题 相反，减轻心脏负荷和减少血管危险因素是主要的治疗方法。 战略。未来的战略应该超越预防和治疗，转向恢复、再生和 功能心脏组织的替换。以下几种机制面临着两个根本挑战 潜在的心肌细胞更新(内源性心肌细胞、常驻祖细胞或 心外祖细胞，包括骨髓源性或多能细胞)：足够数量的 在各种疾病中，是否会产生新的心肌细胞来有意义地改善心功能？而且由于 成年哺乳动物的大多数组织仍处于有丝分裂后，以最大限度地减少细胞转化为 不受控制的增殖性肿瘤状态，能否发现心肌细胞特有的增殖机制 被操纵了吗？目前尚不清楚已报道的心肌细胞增殖机制是否同时符合这两种机制。 挑战。我们最近开发了一种新的平台，用于在 功能性人心肌细胞是由iPS细胞成熟而来的。我们的创新是一种没有假设的、表现型的 旨在发现先前未知的心肌细胞选择性启动子的筛选级联反应 心肌细胞增殖。我们的假设是，这种方法最终会产生类似毒品的起点 用于未来治疗心血管疾病的药物。一次HTS分析已经在一个 384-Well格式，作为分析准备情况的证明，已经筛选了12,000个化合物(Z‘- 系数为0.4)。识别了来自飞行员屏幕的多个命中，并在集中确认和验证 反应实验。已经开发了一系列下游测试来建立关键路径测试 漏斗。从试点筛选中鉴定出的几种化合物专门促进心肌细胞增殖 抗成纤维细胞增殖，并进一步促进了新生儿和成人的体外增殖 分离的大鼠心肌细胞。这项建议建立在申请者的数据基础上，该团队来自 SBP(Larson博士和Colas博士)，具有该领域的基础生物学和药物发现专业知识，并可接触到所有 必要的技术。这项提议的总体目标是产生化学生物学研究工具和 新药的起点。由于关键路径分析都已到位，我们预计我们可以迅速 获得这样的探针分子，并开始探索它们的活性。除了这笔赠款之外，我们未来的计划是 最终确定它们对Hit-to-Lead活动的适用性，开始在体内评估先导化合物 动物模型和最终的病人，并确定他们的细胞作用机制。该产品是 本申请中建议的工作将作为提交Hit-to-Lead(HTL)拨款的初步数据 和父母R01的资助申请，以寻求对其机制的基本生物学理解。

TITLE: Discovery of small molecule promoters of cardiomyocyte proliferation to restore cardiac performance in disease PROJECT SUMMARY Cardiovascular disease is the leading cause of mortality in developed and developing nations. Existing CVD pharmacotherapy options do not treat any foundational problems in the myocardium and its constituent cardiomyocytes; rather, unloading the heart and reducing vascular risk factors are the primary therapeutic strategies. Future strategies should move beyond prevention and treatment to restoration, regeneration, and replacement of functional cardiac tissue. Two fundamental challenges face the several mechanisms of potential cardiomyocyte renewal (proliferation of endogenous cardiomyocytes, resident progenitor cells, or extracardiac progenitor cells including bone-marrow derived or pluripotent cells): can a sufficient number of new cardiomyocytes be generated to meaningfully improve cardiac function in various diseases? And since most tissues in adult mammals remain post-mitotic to minimize the risk of cellular transformation into an uncontrolled proliferating neoplastic state, can cardiomyocyte-specific proliferation mechanisms be discovered and manipulated? It is unclear that the reported mechanisms of cardiomyocyte proliferation meet both of these challenges. We have recently developed a novel platform to conduct high throughput screening (HTS) on functional human cardiomyocytes matured from iPS cells. Our innovation is a hypothesis-free, phenotypic screening cascade designed to discover previously unknown, cardiomyocyte selective promoters of cardiomyocyte proliferation. Our hypothesis is this approach will ultimately generation 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, 12,000 compounds have been screened (Z’- factor of 0.4). Multiple hits from pilot screens were identified and were confirmed and validated in concentration response experiments. A battery of downstream assays has been developed to establish a critical path-testing funnel. Several compounds identified from the pilot screen specifically promoted cardiomyocyte proliferation versus fibroblast proliferation, and furthermore promoted ex vivo proliferation in both neonatal and adult cardiomyocytes isolated from rats. This proposal builds on data from the applicants, an established team from SBP (Drs. Larson and Colas) 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 the 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 beyond this grant are to ultimately determine their 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. The product of the work proposed in this application will serve as preliminary data for both hit-to-lead (HTL) grant submissions and parent R01 grant submissions to pursue basic biology understanding of their mechanisms.