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.

项目摘要 我们的长期目标是开发针对心脏肌质网（SR）CA释放通道的药物 （Ryanodine接收器，RYR2）用于心力衰竭（HF）和心律不齐治疗。 RYR2 CA版本是关键参与者 调节心脏收缩，电生理学，能量和信号传导。异常高的舒张期SR CA 通过RYR2泄漏，并减少了SR CA的吸收，共同减少SR CA含量并提升舒张期[CA] I， 收缩期和舒张期HF的标志。不适当的定时SR CA泄漏也是心律失常。那 RYR2被广泛认为是一个分子靶标的，具有HF的优异治疗潜力和一些 心律不齐。实际上，一些重新利用的药物为此概念提供了原理证明。大大加速 发现针对RYR2泄漏的药物，我们提出了第一个高通量筛查（HTS）方法 使用我们建立的基于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敏感的两个表型特征有关 构象 - 减少钙调蛋白（CAM）结合和生物传感器肽（DPC10）的结合增加。我们 发现SR CA泄漏可以通过强制CAM结合或Dantrolene（用于急性RYR1的药物）逆转 恶性高温泄漏）。 Dantrolene不适合长期使用，因此我们寻求还原的新型药物 正常CAM和DPC10亲和力（RYR构象），从而抑制病理SR CA泄漏。 我们已经建立了基于FRET的CAM和DPC10结合与RYR2的直接测定，以及一种新颖 荧光寿命板阅读器可以将这些FRET工具转换为RYR的超敏化测定法 以HTS格式的构象和与结合伙伴的相互作用。飞行员屏幕的结果证明了 我们被毒死以开展一项明确的药物发现运动，以检测病理生理RYR2 构象并识别恢复正常RYR2构象和功能的化合物，从而翻译 我们对疗法的机械研究。识别此HTS平台和医疗材料的铅化合物 类似物的化学发展将集中在测量RYR活性的次级筛选中 患者衍生的IPSC心肌细胞中的SR膜以及细胞毒性和CA泄漏 衍生的成年心室肌细胞（正常和HF）。可行性是通过：（1）强大而敏感的货 专门解决RYR结构变化的系统，（2）证明了应用此FRET系统的经验 对于RYR1靶向HTS，（3）一种新型的高精度FLT-PR，（4）来自平行假设的功能见解 驱动的机械心肌和动物研究，以及（5）MPI和合作者的顶级团队。中央 假设 - 将CAM和DPC10与RYR2的结合是RYR2病理的关键标记 - 将在 以下具体目的：（1）屏幕集合50k-350k化合物，（2）确定对 RYR2介导的对照和HF肌细胞中的钙泄漏。结果将包括新的HTS分析，一个模型 大规模的HTS活动以及可能发展为新药或RYR2问题的新颖化合物。

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