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

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，以发现用于治疗心力衰竭（HF）的新药，心力衰竭是全球发病和死亡的主要原因。在HF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来源是肌浆网Ca-ATP酶（SERCA 2a）活性下降，其受受磷蛋白（PLB）的调节，PLB是一种抑制SERCA的膜蛋白。二十年来，主要的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，以直接靶向SERCA-PLB相互作用，在优化的测定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，提供不够的精度，导致假阳性和阴性率高。荧光寿命检测可以解决这个问题，但市售的寿命板读数器对于高通量筛选来说太慢。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，并将其纳入新型酶标仪中。该仪器以与最佳荧光强度读取器相同的速率扫描，但精度和分辨率至少提高了一个数量级。因此，初始筛选的有用信息内容得到了显著改善，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筛选，以识别特异性激活心脏中Ca转运的化合物。 目的：（1）优化和验证SERCA-PLB FRET方法在荧光寿命酶标仪上的高通量筛选。(2)在50 K化合物文库上进行（1）中优化的筛选。(3)对（2）中选择的命中进行二级功能测定，为将来的药物研究做准备。 化学和动物实验。该项目是新颖的和探索性的，具有高风险平衡对高影响的潜力，不仅因为新的仪器，而且因为我们采用了新的分子结构，涉及重组膜和活细胞中的整合膜蛋白，以前从未用于高通量筛选。该项目的高度意义部分源于这里发现的化合物的潜力，以革命性地治疗HF，以及与Ca稳态相关的其他疾病（例如，癌症、糖尿病、肌肉萎缩症）。如果我们的方法为基于荧光的药物发现建立了一个新的生物物理范式，那么将会产生更大的影响。 公共卫生相关性：我们将使用我们的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，这是全球住院和死亡的主要原因。这项工作也可能有助于开发其他影响钙转运的疾病的药物，如癌症，糖尿病和肌肉萎缩症。事实上，我们的目标是在药物发现方面进行一场革命，这场革命应该会影响到医学的所有领域。

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.