Chemical Approaches to Rescue Human Mitochondrial Disease Mutations

摘要 线粒体编码的DNA或核编码的线粒体基因的突变可导致 线粒体蛋白的功能表型。线粒体疾病(MD)是一组异质性的 具有几种不同突变的疾病，导致不同的器官表型。一般说来，线粒体 疾病的特点是高能量需求的器官功能减弱，如心脏， 骨骼肌和大脑。此外，患有MD的患者通常患有牙病，需要 独一无二的管理。许多麻醉药抑制线粒体功能，这对 MD患者的治疗。MD患者可发展为进行性呼吸衰竭和乳酸血症 酸中毒，麻醉会加重酸中毒。除了牙科治疗方面的担忧外， MD患者的骨健康状况较差，如骨量减少和骨质疏松。 这项拟议的研究旨在发现可以作为药物靶点的新的基因靶点，以便 恢复携带线粒体突变的细胞的功能和存活。高通量小分子 线粒体脑肌病、乳酸酸中毒、 并进行了中风样发作(MELAS)综合征的研究，以发现导致中风样发作增加的化合物 细胞在营养缺乏的条件下存活。强力霉素被确定为最受欢迎的药物之一。 有趣的是，在Rieske(Complex)上的高通量小分子筛选中也发现了多西环素 三)突变成纤维细胞和ND1(复合体I)突变胞质，采用与MELAS相同的细胞存活试验 屏幕上。在三个独立的小分子生存筛查中，只有两个化合物家族 被确认为阳性的药物：针对线粒体核糖体的抗生素和mTOR抑制剂。MTOR 抑制素此前已在线粒体疾病中进行了研究，因此，本项目将重点关注 多西环素治疗线粒体疾病的疗效。基于跨单元的常见屏幕点击量 LINES，我假设多西环素能够促进线粒体疾病的存活，而不是遗传的 突变。此外，我认为这是通过减少线粒体蛋白质的合成、调节而发生的 应激反应因子，以及细胞死亡途径的抑制。 计划中的实验将首先确定多西环素促进细胞存活的机制。 在线粒体突变的细胞中。Ndufs4基因敲除小鼠，一种成熟的线粒体突变小鼠 具有神经元变性强、寿命短的患者，将用多西环素治疗，以评估其疗效。 多西环素在体内。这项拟议的工作将为新的治疗策略奠定基础，使MD受益 病人。

Abstract Mutations in the mitochondrial encoded DNA or in the nuclear encoded mitochondrial genes can lead to loss of function phenotypes in mitochondrial proteins. Mitochondrial diseases (MD) are a heterogeneous group of disorders with several different mutations leading to a variety of organ phenotypes. In general, mitochondrial diseases are characterized by diminished function of organs with high energetic demands, such as the heart, skeletal muscle, and brain. Additionally, patients with MDs often present with dental disease that requires unique management. Many anesthetic drugs suppress mitochondrial function, which presents a challenge in the treatment of MD patients. Patients with MD can develop progressive respiratory failure and lactic acidosis, which are exacerbated with anesthesia. In addition to dental treatment concerns, manifestations of poor bone heath; such as osteopenia and osteoporosis, have been observed in MD patients. The proposed study aims to discover novel genetic targets that can be pharmaceutically targeted in order to restore function and survival to cells carrying a mitochondrial mutation. A high-throughput small molecule screen on a trans-mitochondrial hybrid (cybrid) model of mitochondrial encephalomyopathy, lactic-acidosis, and stroke-like episodes (MELAS) syndrome was performed to discover compounds that lead to an increase in cell survival in conditions of nutrient deprivation. Doxycycline was identified amongst the top hits. Interestingly, doxycycline was also identified in a high-throughput small molecule screen on Rieske (complex III) mutant fibroblasts and ND1 (complex I) mutant cybrids using the same cell survival assay as the MELAS screen. Across the three independent small molecule survival screens, only two families of compounds were identified as positive hits: antibiotics targeting the mitochondrial ribosome and mTOR inhibitors. mTOR inhibiton has been previously studied in mitochondrial disease, therefore, this project will focus on the efficacy of doxycycline as a therapy in mitochondrial disease. Based off the common screen hits across cell lines, I hypothesize that doxycycline is able promote survival in mitochondrial disease independent of genetic mutation. Further, I believe this is occurring through decreases in mitochondrial protein synthesis, modulation of stress response factors, and inhibition of the cell death pathway. The proposed experiments will first aim to identify the mechanism by which doxycycline promotes cell survival in cells with mitochondrial mutations. Ndufs4 knockout mice, a well-established mitochondrial mutant mouse with strong neuronal deterioration and short lifespan, will be treated with doxycycline to evaluate the efficacy of doxycycline in vivo. The proposed work will lay the groundwork for a novel therapeutic strategy to benefit MD patients.