High-throughput screen to discover SERCA activators for heart failure therapy

高通量筛选发现用于心力衰竭治疗的 SERCA 激活剂

基本信息

批准号：
8448939
负责人：
David D Thomas
金额：
$ 22.8万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-15 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8448939
关键词：
ATP phosphohydrolase Affect Animal Testing Animals Area Basic Science Biological Assay Ca(2+)-Transporting ATPase Calcium Cardiac Cardiac Myocytes Cells Cessation of life Chemicals Complex Data Detection Diabetes Mellitus Disease Effectiveness Equilibrium Excision Family suidae Fluorescence Fluorescence Resonance Energy Transfer Functional disorder Future Goals Heart Heart failure Homeostasis Hospitalization Human Integral Membrane Protein Laboratories Lead Libraries Life Link Lipids Malignant Neoplasms Measurement Measures Medicine Membrane Membrane Proteins Molecular Molecular Structure Morbidity - disease rate Muscle Muscular Dystrophies Myocardium Noise Performance Pharmaceutical Chemistry Pharmaceutical Preparations Play Preparation Problem Solving Procedures Process Protein Isoforms Rattus Reader Reagent Relative (related person)Relaxation Research Personnel Resolution Rest SERCA2a Sampling Sarcoplasm Sarcoplasmic Reticulum Scanning Series Solutions Source Speed Structure System Technology Testing Time Ventricular Work base drug development drug discovery experience high risk high throughput screening improved in vitro Assay innovation instrument instrumentation new technology novel phospholamban reconstitution small molecule stem

项目摘要

DESCRIPTION (provided by applicant): Our goal is to develop novel fluorescence technology, reagents, and concepts, in order to discover new drugs for the treatment of heart failure (HF), the leading cause of morbidity and death worldwide. In HF, a key dysfunction is in Ca transport, which is needed to relax the muscle after each heart beat. A key source of this deficiency is declining activity of the sarcoplasmic reticulum Ca-ATPase (SERCA2a), which is regulated by phospholamban (PLB), a membrane protein that inhibits SERCA. For two decades, major drug companies have tried to develop small-molecule activators of SERCA or inactivators of PLB, but have failed. In this project, we introduce a series of technical and conceptual innovations to solve this problem. We seek to discover small-molecule compounds that disrupt the inhibitory SERCA-PLB interaction. Our approach is to conduct a high-throughput screen using fluorescence resonance energy transfer (FRET) to directly target the SERCA-PLB interaction, in an assay optimized to detect subtle allosteric structural changes required to disrupt inhibition. Based on a pilot screen, we conclude that conventional plate-readers, which measure fluorescence intensity, provide insufficient precision, resulting in high rates of false positives and negatives. Fluorescence lifetime detection could solve this problem, but commercially available lifetime plate readers have been too slow for high-throughput screening. Therefore, we have developed new technology for high- throughput fluorescence lifetime detection, 105 times faster than existing technology, and incorporated it into a novel plate reader. This instrument scans at the same rate as the best fluorescence intensity reader, but precision and resolution are increased by at least an order of magnitude. As a result, the useful information content of the initial screen is substantially improved, dramatically enhancing the speed and effectiveness of drug discovery. This revolutionary technology enables us to conduct a high-throughput fluorescence lifetime screen of the SERCA-PLB complex, to identify compounds that specifically activate Ca transport in the heart. Aims: (1) Optimize and validate the SERCA-PLB FRET assay for high throughput screening in a fluorescence lifetime plate reader. (2) Conduct the screen optimized in (1) on a 50K-compound library. (3) Perform secondary functional assays on hits selected in (2), in preparation for future efforts in medicinal chemistry and animal testing. This project is novel and exploratory, with high risk balancing against the potential for high impact, not only because of the new instrumentation, but also because we employ new classes of molecular constructs, involving integral membrane proteins in both reconstituted membranes and in live cells, never before used in a high-throughput screen. This project's high significance stems in part from the potential of compounds discovered here to revolutionize therapies for HF, as well as for other disorders linked to Ca homeostasis (e.g., cancer, diabetes, muscular dystrophy). Even greater impact will ensue if our approach establishes a new biophysical paradigm for fluorescence-based drug discovery. PUBLIC HEALTH RELEVANCE: We will use our novel fluorescence detection technology to discover drugs that specifically activate the cardiac calcium pump to help cure heart failure, the leading cause of hospitalization and death worldwide. This work is likely to also help develop drugs for other disorders affecting calcium transport, such as cancer, diabetes, and muscular dystrophy. Indeed, our goal is a revolution in drug discovery that should affect all areas of medicine.

描述（由申请人提供）：我们的目标是开发新型荧光技术、试剂和概念，以发现治疗心力衰竭（HF）的新药，心力衰竭是全世界发病和死亡的主要原因。在心力衰竭中，一个关键的功能障碍是钙转运，这是每次心跳后放松肌肉所必需的。这种缺陷的一个关键根源是肌浆网 Ca-ATP 酶 (SERCA2a) 活性下降，该酶受磷蛋白 (PLB)（一种抑制 SERCA 的膜蛋白）调节。二十年来，各大制药公司一直试图开发SERCA的小分子激活剂或PLB的灭活剂，但都失败了。在这个项目中，我们引入了一系列技术和概念创新来解决这个问题。我们寻求发现破坏抑制性 SERCA-PLB 相互作用的小分子化合物。我们的方法是使用荧光共振能量转移 (FRET) 进行高通量筛选，直接靶向 SERCA-PLB 相互作用，在优化的检测中检测破坏抑制所需的微妙变构结构变化。根据试点屏幕，我们得出结论，测量荧光强度的传统读板仪精度不够，导致假阳性和假阴性率很高。荧光寿命检测可以解决这个问题，但市售的寿命酶标仪对于高通量筛选来说速度太慢。因此，我们开发了用于高通量荧光寿命检测的新技术，比现有技术快 105 倍，并将其整合到新型酶标仪中。该仪器的扫描速率与最佳荧光强度读取器相同，但精度和分辨率至少提高了一个数量级。结果，初始筛选的有用信息内容得到了显着改善，显着提高了药物发现的速度和有效性。这项革命性的技术使我们能够对 SERCA-PLB 复合物进行高通量荧光寿命筛选，以识别特异性激活心脏中 Ca2+ 转运的化合物。目标：(1) 优化并验证 SERCA-PLB FRET 测定，以在荧光寿命板读数器中进行高通量筛选。 (2) 在 50K 化合物库上进行 (1) 中优化的筛选。 (3) 对(2)中选择的命中进行二次功能测定，为未来的药物研究工作做好准备化学和动物测试。这个项目是新颖的和探索性的，具有高风险与高影响潜力的平衡，不仅因为新的仪器，而且因为我们采用了新型分子结构，涉及重构膜和活细胞中的完整膜蛋白，以前从未在高通量筛选中使用过。该项目的重要意义部分源于这里发现的化合物可能彻底改变心力衰竭以及与钙稳态相关的其他疾病（例如癌症、糖尿病、肌营养不良）的治疗方法。如果我们的方法为基于荧光的药物发现建立新的生物物理范例，将会产生更大的影响。公共健康相关性：我们将利用新型荧光检测技术来发现专门激活心脏钙泵的药物，以帮助治疗心力衰竭，心力衰竭是全球住院和死亡的主要原因。这项工作还可能有助于开发治疗影响钙转运的其他疾病的药物，例如癌症、糖尿病和肌营养不良症。事实上，我们的目标是药物发现的一场革命，这应该影响医学的所有领域。