Rational Design from Cryo-EM Structures of High-Affinity Ryanodine Receptor Ligands Based on Natural Peptides

基于天然肽的高亲和力兰尼定受体配体的冷冻电镜结构的合理设计

基本信息

批准号：
10729564
负责人：
Hector H Valdivia
金额：
$ 66.4万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-10 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10729564
关键词：
Adrenergic Agents Affect Affinity Agonist Amino Acids Animal Model Animals Area Arrhythmia Arrhythmogenic Right Ventricular Dysplasia Atrial Fibrillation Binding Biological Assay Biology Cardiac Cardiac Myocytes Cardiomyopathies Catecholaminergic Polymorphic Ventricular Tachycardia Cells Child Complex Coupling Cryoelectron Microscopy DNA Sequence Alteration Development Diastole Disease Family Functional disorder Genetic Transcription Goals Heart Heart Diseases Heart failure Hyperactivity Hypertrophic Cardiomyopathy Individual Ions Knock-in Label Ligands Link Marketing Mediating Membrane Modeling Molecular Mus Muscle Cells Muscle Fibers Mutation Oryctolagus cuniculus Pathologic Penetration Peptides Perfusion Permeability Pharmaceutical Preparations Pharmacotherapy Phase Play Process Property Rationalization Receptor Gene Role RyR2 Ryanodine Receptor Calcium Release Channel Sarcoplasmic Reticulum Scorpions Series Site Specificity Structure Sudden Death Syndrome Techniques Testing Therapeutic Transmembrane Domain Venoms Ventricular Ventricular Tachycardia Vestibule analog design effective therapy gain of function mutation insight member mutant nanomolar novel pharmacologic prevent programs rational design stem transcription factor young adult

项目摘要

SUMMARY Ryanodine receptors (RyRs), are the Ca2+ release channels of sarcoplasmic reticulum (SR) that play an essential role in excitaton-contraction coupling of cardiac and skeletal muscle cells. Mutations in the cardiac RyR gene (RYR2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia and sudden death. CPVT is clearly an arrhythmogenic disorder stemming from intracellular Ca2+ mishandling caused by RyR2 dysfunction. As such, insights gained from CPVT syndromes benefit our mechanistic understanding of other cardiomyopathies where RyR2-linked Ca2+ mishandling plays a pivotal role. RyR2 dysfunction brings about deleterious effects in the heart by inducing SR Ca2+ “leak” and/or spontaneous Ca2+ release (SCR), a sudden, diastolic Ca2+ bolous of critical mass that triggers arrhythmic activity. Thus, reigning in RyR2 hyperactivity to mitigate SR Ca2+ “leak” and avoid SCR is a primary goal of therapeutic regimes for major cardiomyopathies. A group of globular peptides termed calcins target RyRs with high affinity and specificity. Imperacalcin (IpCa), the founding member of the calcin family, engages RyRs by entering through their wide vestibule and binding with exquisite precision to a site deep in the cytosolic “cap” of the channel, adjacent to the transmembrane region, acting as a wedge that “pushes” laterally the transmembrane helices that line the channel pore, causing it to open and giving rise to a long-lived subconductance state. In animal models of CPVT, IpCa penetrates the external membrane of ventricular myocytes and induces a partial depletion of SR Ca2+, thus preventing SCR and in effect relieving the adrenergically-mediated Ca2+ overload that triggers Ca2+-dependent arrhythmias. Thus, IpCa behaves naturally as an agonist of RyRs, with anti-arrhytmic effect in cardiomyopathies where SCR is the primary trigger of arrhythmias, and by rational design of IpCa analogs capable of closing the natural “grooves” it creates with RyRs, it has the tangible potential to become the first high-affinity RyR blocker that may prevent SR Ca2+ “leak”. Using cryo-EM structures of calcin-RyR complexes and a series of functional assays, aim 1 will determine the structural domains of calcins that allow them to bind to RyRs with exquisite affinity and specificity, and to generate calcin analogs capable of blocking RyR ion conduction. Aim 2 will use CPVT mouse lines that readily present SCR during sympathetic stimulation and a novel rabbit CPVT model that presents constitutive SR Ca2+ leak and pathological cardiac remodeling to determine whether native and mutant calcins are capable of preventing RyR dysfunction-triggered arrhythmias. Our multi-disciplnary program, with well-defined deliverables and milestones and led by two PIs experts in structural (Van Petegem) and functional (Valdivia) biology of RyRs, will produce a novel paradigm for the treatment of Ca2+-dependent arrhythmias, an area severely underserved by existent pharmacotherapy.

概括 Ryanodine 受体 (RyRs) 是肌浆网 (SR) 的 Ca2+ 释放通道，在心脏 RyR 基因突变在心肌细胞和骨骼肌细胞的兴奋-收缩耦合中发挥重要作用。 (RYR2) 与儿茶酚胺能多形性室性心动过速 (CPVT) 相关，CPVT 是一种致心律失常以肾上腺素介导的室性心动过速和猝死为特征的综合征。 CPVT 显然是一种致心律失常性疾病，源于 RyR2 引起的细胞内 Ca2+ 处理不当因此，从 CPVT 综合征中获得的见解有利于我们对其他疾病的机制理解。 RyR2 相关的 Ca2+ 处理不当在导致 RyR2 功能障碍中发挥关键作用的心肌病。通过诱导 SR Ca2+“泄漏”和/或自发 Ca2+ 释放 (SCR)、突然的舒张期对心脏产生有害影响触发心律失常活动的临界质量的 Ca2+ 丸剂因此，控制 RyR2 过度活跃以减轻 SR Ca2+。 “渗漏”和避免 SCR 是主要球状心肌病治疗方案的主要目标。称为钙素的肽以高亲和力和特异性靶向 RyR，而 Imperacalcin (IpCa) 是 RyR 的创始成员。 calcin 家族通过进入 RyR 的宽阔前庭并以精致的精度结合到某个位置来吸引 RyR 位于通道胞质“帽”深处，邻近跨膜区域，充当“推动”的楔子横向排列通道孔的跨膜螺旋，使其打开并产生长寿命的在 CPVT 动物模型中，IpCa 穿透心室肌细胞的外膜。并诱导 SR Ca2+ 部分耗尽，从而防止 SCR 并有效缓解肾上腺素介导的 Ca2+ 过载会引发 Ca2+ 依赖性心律失常，因此，IpCa 自然地表现为 RyRs 激动剂。在 SCR 是心律失常的主要触发因素的心肌病中具有抗心律失常作用，并通过合理设计 IpCa 类似物能够封闭它与 RyRs 产生的自然“凹槽”，它有可能成为第一个高亲和力 RyR 阻断剂，可利用 calcin-RyR 复合物的冷冻电镜结构防止 SR Ca2+“泄漏”。和一系列功能测定，目标 1 将确定钙素的结构域，使它们能够结合 RyRs具有精湛的亲和力和特异性，并生成能够阻断RyR离子传导的钙素类似物。目标 2 将使用在交感神经刺激期间容易呈现 SCR 的 CPVT 小鼠系和新型兔 CPVT 模型呈现组成型 SR Ca2+ 渗漏和病理性心脏重塑，以确定天然和突变型钙素能够预防 RyR 功能障碍引发的心律失常。明确的可交付成果和里程碑，由结构（Van Petegem）和功能领域的两位 PI 专家领导 (Valdivia) RyRs 的生物学，将为治疗 Ca2+ 依赖性心律失常（一个领域）提供一种新的范例存在主义药物疗法严重缺乏服务。