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

高通量筛选发现用于心力衰竭治疗的漏性 RyR2 新型抑制剂

• 批准号: 10064096
• 负责人: Donald M Bers
• 金额: $ 75.42万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-15 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10064096
• 关键词: Academia; Action Potentials; Acute; Adult; Adverse effects; Affinity; American; Animal Model; Animals; Arrhythmia; Back; Basic Science; Binding; Biochemical; Biological Assay; Biosensor; Calcium; Calmodulin; Cardiac; Cardiac Myocytes; Catecholaminergic Polymorphic Ventricular Tachycardia; Cell model; Chemicals; Chronic; Clinical; Collection; Confocal Microscopy; Coupled; Critical Pathways; Dantrolene; Data; Defect; Detection; Development; Diastole; Diastolic heart failure; Drug Incompatibility; Drug Targeting; Drug usage; Electrophysiology (science); Ensure; FKBP1B gene; Family suidae; Fluorescence; Fluorescence Resonance Energy Transfer; Future; Goals; Health; Heart; Heart Diseases; Heart failure; Human; Industrialization; Industry; Label; Lead; Letters; Libraries; Link; Malignant hyperpyrexia due to anesthesia; Measures; Mediating; Membrane; Methods; Modeling; Molecular; Molecular Conformation; Molecular Target; Muscle Cells; Outcome; Oxidative Stress; Pathologic; Pathology; Patients; Peptides; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phenotype; Proteins; Reader; Research; RyR1; RyR2; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Signal Transduction; Skeletal Muscle; Structure; System; Tacrolimus Binding Proteins; Technology; Testing; Therapeutic; Toxic effect; Translating; Translations; Treatment Failure; Ventricular; Vesicle; analog; base; calmodulin-dependent protein kinase II; drug development; drug discovery; drug repurposing; experience; high throughput screening; in vivo; induced pluripotent stem cell; inhibitor/antagonist; insight; notch protein; novel; novel therapeutics; screening; tool; uptake

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.

项目概要 我们的长期目标是开发针对心脏肌浆网 (SR) Ca 释放通道的药物 （兰尼碱受体，RyR2）用于心力衰竭 (HF) 和心律失常治疗，RyR2 Ca 释放在治疗中发挥着关键作用。 调节心脏收缩、电生理学、能量学和信号传导。 通过 RyR2“泄漏”，并减少 SR Ca 摄取，共同降低 SR Ca 含量并升高舒张压 [Ca]i， 收缩期和舒张期心力衰竭的特征是，不适当的 SR 钙漏也会导致心律失常。 RyR2 被广泛认为是对心力衰竭和某些疾病具有优异治疗潜力的分子靶点。 事实上，一些重新利用的药物为这一概念提供了原理验证，以大大加速这一过程。 发现针对 RyR2 泄漏的药物，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。 病理相关的 RyR 泄漏与对 RyR 敏感的两种表型特征相关 构象 – 钙调蛋白 (CaM) 结合减少，生物传感器肽 (DPc10) 结合增加。 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 RyR1 的药物）来逆转 恶性高热中的渗漏）。丹曲林不适合长期使用，因此我们寻求能够恢复的新药物。 正常的 CaM 和 DPc10 亲和力（RyR 构象），从而抑制病理性 SR Ca 渗漏。 我们已经建立了基于 FRET 的直接 CaM 和 DPc10 与 RyR2 结合的测定法，以及一种新颖的方法 荧光寿命读板机能够将这些 FRET 工具转化为 RyR 的超灵敏检测 中试筛选结果以 HTS 格式展示了构象和与结合伙伴的相互作用。 我们准备开展明确的药物发现活动来检测病理生理学 RyR2 构象并识别恢复正常 RyR2 构象和功能的化合物，从而翻译 我们从该 HTS 平台和药物中鉴定先导化合物的机制研究。 类似物的化学开发将集中于测量 RyR 活性的二次筛选 SR 膜、患者来源的 iPSC 心肌细胞和动物体内的细胞毒性和 Ca 渗漏 衍生的成体心室肌细胞（正常和 HF）通过以下方式确保可行性：（1）稳健且灵敏的 FRET。 系统专门解决 RyR 结构变化，(2) 展示了应用该 FRET 系统的经验 对于 RyR1 靶向 HTS，（3）一种新颖的高精度 FLT-PR，（4）来自并行假设的功能洞察 - 驱动的机械肌细胞和动物研究，以及（5）一流的 MPI 和合作者团队。 假设 – CaM 和 DPc10 与 RyR2 的结合是 RyR2 病理学的关键标记 – 将在 以下具体目标：(1) 筛选 50k-350k 种化合物的集合，以及 (2) 确定 Hit 对 RyR2 介导的对照和 HF 肌细胞中的钙渗漏结果将包括新的 HTS 测定，这是一种模型。 大规模的 HTS 活动，以及可能开发成新药或 RyR2 探针的新化合物。

项目成果

Patient-Specific Induced Pluripotent Stem Cells Implicate Intrinsic Impaired Contractility in Hypoplastic Left Heart Syndrome.

• DOI: 10.1161/circulationaha.119.045317
• 发表时间: 2020-10-20
• 期刊: Circulation
• 影响因子: 37.8
• 作者: Paige SL;Galdos FX;Lee S;Chin ET;Ranjbarvaziri S;Feyen DAM;Darsha AK;Xu S;Ryan JA;Beck AL;Qureshi MY;Miao Y;Gu M;Bernstein D;Nelson TJ;Mercola M;Rabinovitch M;Ashley EA;Parikh VN;Wu SM
• 通讯作者: Wu SM

Regulation of cardiac calcium signaling by newly identified calcium pump modulators.

新发现的钙泵调节剂对心脏钙信号的调节。

• DOI: 10.1016/j.bbrc.2023.149136
• 发表时间: 2023
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: Bovo,Elisa;Rebbeck,RobynT;Roopnarine,Osha;Cornea,RazvanL;Thomas,DavidD;Zima,AlekseyV
• 通讯作者: Zima,AlekseyV

Human iPSC modeling of heart disease for drug development.

• DOI: 10.1016/j.chembiol.2021.02.016
• 发表时间: 2021-03-18
• 期刊: Cell chemical biology
• 影响因子: 8.6
• 作者: Hnatiuk AP;Briganti F;Staudt DW;Mercola M
• 通讯作者: Mercola M

ent-Verticilide B1 Inhibits Type 2 Ryanodine Receptor Channels and is Antiarrhythmic in Casq2 -/- Mice.

ent-Verticilide B1 抑制 Casq2 -/- 小鼠的 2 型 Ryanodine 受体通道并具有抗心律失常作用。

• DOI: 10.1124/molpharm.123.000752
• 发表时间: 2024
• 期刊: Molecular pharmacology
• 影响因子: 3.6
• 作者: Gochman,Aaron;Do,TriQ;Kim,Kyungsoo;Schwarz,JacobA;Thorpe,MadelaineP;Blackwell,DanielJ;Ritschel,PaxtonA;Smith,AbigailN;Rebbeck,RobynT;Akers,WendellS;Cornea,RazvanL;Laver,DerekR;Johnston,JeffreyN;Knollmann,BjornC
• 通讯作者: Knollmann,BjornC

New N-aryl-N-alkyl-thiophene-2-carboxamide compound enhances intracellular Ca2+ dynamics by increasing SERCA2a Ca2+ pumping.

新型 N-芳基-N-烷基-噻吩-2-甲酰胺化合物通过增加 SERCA2a Ca2 泵送来增强细胞内 Ca2 动力学。

• DOI: 10.1016/j.bpj.2022.12.002
• 发表时间: 2023
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Nikolaienko,Roman;Bovo,Elisa;Yuen,SamanthaL;Treinen,LevyM;Berg,Kaja;Aldrich,CourtneyC;Thomas,DavidD;Cornea,RazvanL;Zima,AlekseyV
• 通讯作者: Zima,AlekseyV