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

• 批准号: 8545666
• 负责人: David D Thomas
• 金额: $ 17.96万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 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/8545666
• 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

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.

描述（由申请人提供）：我们的目标是开发新型的荧光技术，试剂和概念，以发现治疗心力衰竭的新药（HF），这是全球发病率和死亡的主要原因。在HF中，CA运输中的关键功能障碍是在每次心脏跳动后放松肌肉所需的。这种缺陷的关键来源是肌浆网ca-ATPase（SERCA2A）的活性下降，该活性受磷团（PLB）调节的膜蛋白，这是一种抑制SERCA的膜蛋白。二十年来，主要制药公司一直试图开发SERCA或PLB灭活剂的小分子激活剂，但失败了。 在这个项目中，我们介绍了一系列技术和概念创新来解决此问题。我们试图发现破坏抑制性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）进行高通量屏幕，以直接针对SERCA-PLB相互作用，以优化的测定，以检测破坏抑制所需的细微变构结构变化。基于试点屏幕，我们得出结论，测量荧光强度的常规板读取器提供了不足的精度，从而导致误报率很高。荧光寿命检测可以解决此问题，但是在高通量筛选中，可商购的寿命读取器太慢了。因此，我们开发了用于高吞吐量荧光寿命检测的新技术，比现有技术快105倍，并将其纳入新的板块读取器中。该仪器以与最佳荧光强度读取器相同的速率进行扫描，但精度和分辨率至少提高了一个数量级。结果，初始屏幕的有用信息内容得到了显着改善，从而大大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屏幕，从而识别专门激活心脏中CA转运的化合物。 目的：（1）优化和验证SERCA-PLB FRET分析，以在荧光寿命读取器中进行高吞吐量筛选。 （2）在50k混合库上（1）中进行优化的屏幕。 （3）对（2）中选择的命中进行辅助功能测定，以准备未来的药用努力 化学和动物测试。该项目是新颖和探索性的，具有高风险与高影响潜力的高风险平衡，这不仅是因为新的仪器，而且还因为我们采用了新的分子构建体，涉及在重建的膜和活细胞中涉及整体膜蛋白和在活细胞中，从来没有在高通量屏幕中使用。该项目的高意义部分源于此处发现的化合物的潜力，以彻底改变HF的疗法以及与CA稳态相关的其他疾病（例如癌症，糖尿病，肌肉营养不良）。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式，那么将会产生更大的影响。