High-Throughput Screens to Discover Novel Inhibitors of Leaky RyR2 for Heart Failure Therapy

项目摘要 我们的长期目标是开发靶向心脏肌浆网（SR）钙释放通道的药物 （ryanodine receptor，RyR 2）用于心力衰竭（HF）和心律失常治疗。RyR 2钙释放是一个关键的球员， 调节心脏收缩、电生理学、能量学和信号传导。舒张期SR Ca异常高 通过RyR 2的“泄漏”和减少的SR Ca摄取，共同减少SR Ca含量和升高舒张[Ca]i， 收缩期和舒张期心力衰竭的特征不适当的SR Ca泄漏时间也是致瘤性的。因此 RyR 2被广泛认为是对HF具有优异治疗潜力的分子靶点，并且一些 心律不齐事实上，一些重新利用的药物为这一概念提供了原理证明。大大加快了 在发现靶向RyR 2泄漏的药物后，我们提出了第一个高通量筛选（HTS）方法 使用我们完善的基于FRET的RyR靶向系统和广泛的支持基础研究。 病理相关RyR泄漏与两种对RyR敏感的表型特征相关 构象降低的钙调蛋白（CaM）结合和增加的生物传感器肽（DPc 10）结合。我们 发现SR Ca泄漏可以通过强制CaM结合或丹曲林（一种用于急性RyR 1的药物）逆转。 恶性高热中泄漏）。丹曲林不适合长期使用，所以我们寻求新的药物， 正常的CaM和DPc 10亲和力（RyR构象），从而抑制病理性SR Ca泄漏。 我们已经建立了直接基于FRET的CaM和DPc 10与RyR 2结合的测定法， 荧光寿命板读取器使这些FRET工具能够翻译成RyR的超灵敏测定 构象和与结合伴侣的相互作用，以HTS格式。试验性筛选的结果表明， 我们准备开展一项明确的药物发现活动， 构象和鉴定恢复正常RyR 2构象和功能的化合物，从而翻译 我们对疗法的机械研究。从该HTS平台和药物中鉴定先导化合物 类似物的化学开发将通过测量RyR活性的二次筛选来集中， SR膜，以及患者来源的iPSC心肌细胞和动物来源的iPSC心肌细胞中的细胞毒性和Ca泄漏。 衍生的成体心室肌细胞（正常和HF）。可行性通过以下方式确保：（1）稳健且灵敏的FRET 系统，以具体解决RyR结构的变化，（2）证明了应用这种FRET系统的经验 对于RyR 1靶向HTS，（3）一种新的高精度FLT-PR，（4）来自平行假设的功能见解- 驱动的机械肌细胞和动物研究，和（5）一流的MPI和合作者团队。中央 假设-CaM和DPc 10与RyR 2结合是RyR 2病理学的关键标志-将在 以下具体目标：（1）筛选50 k-350 k化合物的集合，以及（2）确定对 RyR 2介导的对照和HF心肌细胞的钙渗漏。结果将包括新的HTS检测， 大规模HTS活动，以及可能开发成新药或RyR 2探针的新化合物。

Project Summary Our long-term goal is to develop drugs that target the cardiac sarcoplasmic reticulum (SR) Ca release channel (ryanodine receptor, RyR2) for heart failure (HF) and arrhythmia therapy. RyR2 Ca release is a key player in regulating cardiac contraction, electrophysiology, energetics and signaling. Abnormally high diastolic SR Ca “leak” via RyR2, and reduced SR Ca uptake, conspire to reduce SR Ca content and elevate diastolic [Ca]i, hallmarks of both systolic and diastolic HF. Inappropriately timed SR Ca leak is also arrhythmogenic. Thus RyR2 is widely recognized as a molecular target with excellent therapeutic potential for HF and some arrhythmias. Indeed, some repurposed drugs provide proof-of-principle for this concept. To greatly accelerate discovery of drugs that target the RyR2 leak, we propose the first high-throughput screening (HTS) methods using our well-established FRET-based RyR-targeting system and extensive supporting basic research. Pathology-associated RyR leak is associated with two phenotypic features that are sensitive to RyR conformation – reduced calmodulin (CaM) binding and increased binding of a biosensor peptide (DPc10). We find that SR Ca leak can be reversed by either forced CaM binding or dantrolene (a drug used for acute RyR1 leak in malignant hyperthermia). Dantrolene is unsuitable for chronic use, so we seek novel drugs that restore normal CaM and DPc10 affinity (RyR conformation) and thus inhibit pathological SR Ca leak. We have established direct FRET-based assays of CaM and DPc10 binding to RyR2, and a novel fluorescence lifetime plate-reader enables the translation of these FRET tools into ultrasensitive assays of RyR conformation and interactions with binding partners, in HTS format. Results from pilot screens demonstrate that we are poised to carry out an explicit drug-discovery campaign to detect pathophysiological RyR2 conformations and identify compounds that restore normal RyR2 conformation and function, thus translating our mechanistic research into therapies. Identification of lead compounds from this HTS platform and medicinal chemistry development of analogues will be focused through secondary screens that measure RyR activity in SR membranes, and cellular toxicity and Ca leak in patient-derived iPSC cardiomyocytes and in animal- derived adult ventricular myocytes (normal and HF). Feasibility is ensured by: (1) a robust and sensitive FRET system to specifically resolve RyR structural changes, (2) demonstrated experience applying this FRET system for RyR1-targeted HTS, (3) a novel high-precision FLT-PR, (4) functional insight from parallel hypothesis- driven mechanistic myocyte and animal studies, and (5) top-notch team of MPIs and collaborators. The central hypothesis – that binding of CaM and DPc10 to RyR2 are key markers of RyR2 pathology – will be tested in the following Specific Aims: (1) Screen a collection of 50k-350k compounds, and (2) Determine Hit effects on RyR2-mediated calcium leak in control and HF myocytes. Outcomes will include new HTS assays, a model for large-scale HTS campaigns, and novel compounds that may be developed into new drugs or RyR2 probes.