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

描述(申请人提供)：我们的目标是开发新的荧光技术、试剂和概念，以发现治疗心力衰竭(HF)的新药，心力衰竭是全球发病率和死亡的主要原因。在心力衰竭中，一个关键的功能障碍是钙的运输，这是在每次心跳后放松肌肉所需的。这种缺陷的一个关键来源是肌浆网Ca-ATPase(SERCA2a)活性下降，该酶受磷蛋白(PLB)调节，磷蛋白是一种抑制SERCA的膜蛋白。二十年来，大型制药公司一直试图开发SERCA的小分子激活剂或PLB的灭活剂，但都以失败告终。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。我们试图发现能破坏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，以直接靶向SERCA-PLB相互作用，在一种优化的方法中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，传统的平板阅读器，测量荧光强度，提供的精度不够，导致高的假阳性和假阴性率。荧光寿命检测可以解决这个问题，但商业上可用的寿命板阅读器对于高通量筛选来说太慢了。因此，我们开发了高通量荧光寿命检测的新技术，比现有技术快105倍，并将其集成到新型平板阅读器中。这种仪器的扫描速度与最好的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大幅改进，极大地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复合体进行高通量荧光寿命筛选，以识别特定激活心脏中钙转运的化合物。目的：(1)优化和验证SERCA-PLB FRET方法在荧光寿命平板阅读器中的高通量筛选。(2)在50K化合物库上进行(1)中优化的屏幕。(3)对(2)中选择的HITS进行二次功能分析，为下一步的医学工作做准备 化学和动物实验。这个项目是新颖的和探索性的，高风险与高影响的可能性相平衡，这不仅是因为新的仪器，还因为我们使用了新的分子结构，包括重组膜和活细胞中的完整膜蛋白，这是以前从未在高通量筛选中使用过的。该项目的高度意义部分源于这里发现的化合物有可能对心衰以及其他与钙稳态相关的疾病(如癌症、糖尿病、肌肉营养不良)的治疗产生革命性的影响。如果我们的方法为基于荧光的药物发现建立一个新的生物物理范式，将会产生更大的影响。

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.