Inducers of Mitochondrial Biogenesis

描述（由申请人提供）：该项目的长期目标是鉴定和开发诱导线粒体生物发生的药物。各种各样的急性损伤，如缺血/再灌注（I/R）、药物、毒物和创伤，导致线粒体功能障碍，并导致许多器官（如心、脑、肝和肾）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一个组成部分，如代谢综合征、神经退行性变和衰老。目前还没有有效的治疗方法可以恢复线粒体功能，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组中具有广泛的效用。线粒体生物发生药物的开发一直受到线粒体功能有限的不良细胞模型、具有高假阳性和假阴性率的生物发生替代标志物以及没有中等到高通量筛选方法来测量功能性线粒体生物发生的阻碍。在过去的五年里，我们在肾近端小管细胞（RPTC）的原代培养中研究了线粒体的生物发生，这些细胞在体内保持线粒体功能。最近，我们对96口Seahorse Biosciences呼吸计的RPTC系统进行了调整和优化，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解偶率仅受功能性线粒体数量的限制，因此，FCCP解偶率的增加是线粒体生物发生的功能性生物标志物。使用该方法，我们从DIVERSet的50,000个化合物文库中随机选择了500个化合物进行筛选。我们确定了7个命中，并通过线粒体蛋白表达增加的二级分析（“金标准”）证实。因此，我们可以立即开始使用化合物文库的表型测定来鉴定线粒体生物发生的新诱导剂。我们假设，通过我们的分析筛选一个大的、多样化的化学文库将导致鉴定产生线粒体生物发生的新化学型，从而导致在体内产生线粒体生物发生的新化学实体。在项目的第一期，我们建议筛选DIVERSet 5万复合库。该文库将首先作为五种化合物池（Aim 1）进行筛选，然后进行反卷积到单分子（Aim 2），最后使用线粒体蛋白质和DNA的生化分析验证命中（Aim 3）。在第二阶段，我们将使用硅药效团分析和QSAR来确定化学型，合成类似物以提高活性，并确定最佳类似物在RPTC和小鼠中诱导线粒体生物发生的功效和效力。

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.