Inducers of Mitochondrial Biogenesis

描述(由申请人提供)：该项目的长期目标是识别和开发诱导线粒体生物发生的药物。各种不同的急性损伤，如缺血/再灌注、药物、毒物和创伤，都会导致线粒体功能障碍，并导致许多器官(如心、脑、肝、肾)的细胞损伤和死亡。此外，线粒体功能障碍是许多慢性疾病的组成部分，如代谢综合征、神经退行性变和衰老。目前还没有可以恢复线粒体功能的有效疗法，因此，一种产生线粒体生物发生的药物可能在治疗广泛的疾病方面具有广泛的实用价值。线粒体生物发生药物的开发一直受到以下因素的阻碍：线粒体功能有限的细胞模型差，生物发生的替代标记物具有高的假阳性和假阴性率，以及没有中高通量的筛选方法来衡量线粒体的功能生物发生。在过去的五年里，我们研究了在原代培养的肾近端小管细胞(RPTC)中线粒体的生物发生，这些细胞在体内保持类似线粒体的功能。我们最近对我们的RPTC系统进行了调整和优化，使其适用于96孔的海马生物科学呼吸仪，并使用已知的诱导线粒体生物发生的化学物质验证了该分析。研究发现，这些细胞的最大FCCP解偶联速率仅受功能线粒体数量的限制，因此，FCCP解偶联速率的增加是线粒体生物发生的功能生物标志物。使用这种方法，我们筛选了从DIVERSet 50,000化合物库中随机选择的500个化合物的测试库。我们确定了7个命中，证实了线粒体蛋白表达增加的二次检测，这是“金标准”。因此，我们可以立即开始使用我们的表型分析与化合物文库，以确定新的线粒体生物发生的诱导剂。我们假设，用我们的方法筛选一个大型、多样化的化学文库将导致鉴定出产生线粒体生物发生的新化学类型，从而导致在体内产生线粒体生物发生的新化学实体。在项目的第一阶段，我们建议筛选DIVERSet 50,000化合物文库。文库最初将被筛选为五种化合物的池(目标1)，然后去卷积为单分子(目标2)，最后使用线粒体蛋白质和DNA的生化分析来验证命中(目标3)。在第二阶段，我们将确定用于电子药效团分析和QSAR的化学类型，合成类似物以提高活性，并确定最好的类似物在诱导RPTC和小鼠线粒体生物发生方面的有效性和潜力。) 公共卫生相关性：线粒体功能障碍是急性组织损伤和慢性病的主要因素。目前，在各种急慢性损伤后，还没有有效的治疗方法可以恢复线粒体的功能，以促进细胞和器官的修复/再生。因此，需要研究开发促进线粒体生物发生的新药。

DESCRIPTION (provided by applicant): The long-term goal of this project is to identify and develop drugs that induce mitochondrial biogenesis. A variety of diverse acute insults such as ischemia/reperfusion (I/R), drug, toxicant, and trauma lead to mitochondrial dysfunction and results in cell injury and death in many organs (e.g. heart, brain, liver and kidney). Furthermore, mitochondrial dysfunction is a component of many chronic diseases such as metabolic syndrome and neurodegeneration, and aging. There are no effective therapies that can restore mitochondrial function and, thus, a drug that produces mitochondrial biogenesis may have extensive utility in the treatment of a broad group of diseases. Mitochondrial biogenesis drug development has been hindered by poor cellular models with limited mitochondrial function, surrogate markers of biogenesis that have high false-positive and false negative-rates, and no moderate to high throughput screening assay to measure functional mitochondrial biogenesis. We have studied mitochondrial biogenesis for the past five years in primary cultures of renal proximal tubular cells (RPTC) that maintain in vivo like mitochondrial function. We recently adapted and optimized our RPTC system to the 96-well Seahorse Biosciences respirometer and validated the assay using chemicals known to induce mitochondrial biogenesis. It was found that the maximum FCCP-uncoupled rates in these cells are limited only by the number of functional mitochondria and, thus, increases in the FCCP- uncoupled rates are functional biomarkers of mitochondrial biogenesis. Using this assay we screened a test library of 500 compounds randomly selected from the DIVERSet 50,000-compound library. We identified 7 hits that were confirmed with secondary assays of increased expression of mitochondrial proteins, the "gold standard". Thus, we can start immediately to use our phenotypic assay with compound libraries to identify new inducers of mitochondrial biogenesis. We hypothesize that screening a large, diverse chemical library with our assay will result in the identification of new chemotypes that produce mitochondrial biogenesis, leading to new chemical entities that produce mitochondrial biogenesis in vivo. In Phase I of the project we propose to screen the DIVERSet 50,000 compound library. The library will be initially screened as pools of five compounds (Aim 1) followed by deconvolution to single molecules (Aim 2), and finally validation of the hits using biochemical assays for mitochondrial proteins and DNA (Aim 3). In Phase II, we will identify chemotypes using in silico pharmacophore analyses and QSAR, synthesize analogues to improve activity, and determine efficacy and potency of the best analogues in inducing mitochondrial biogenesis in RPTC and in mice. ) PUBLIC HEALTH RELEVANCE: Mitochondrial dysfunction is a major contributor to acute tissue injuries and chronic diseases. At this time, there are no effective therapies that can restore mitochondrial function to promote cell and organ repair/regeneration after diverse acute and chronic injuries. Thus, research is needed to develop new drugs that promote mitochondrial biogenesis.