Small molecule modulators for mitochondrial protein import

描述（由申请人提供）：线粒体生物发生缺陷导致广泛的疾病，包括神经变性、中风、心肌梗死、缺血和癌症;然而，纠正这些疾病的疗法并不容易获得。我们建议在分子库生产中心网络（MLPCN）中进行高通量筛选，该筛选与在较小的筛选规模上已经成功鉴定线粒体TIM22蛋白输入途径的抑制剂相同，该途径是模式生物酿酒酵母中内膜蛋白组装所需的。具体而言，我们已经设计了一种基于生长的测定，使用酵母温度敏感突变体，由于有缺陷的输入系统，该突变体在高温下生长受到损害。在筛选中，我们选择在正常允许生长的温度下对突变株具有合成致死性的小分子。使用该测定，我们已经鉴定了特异性靶向酵母线粒体中的TIM 22输入途径的小分子。该提案的目的是（1）鉴定靶向TIM 22输入途径并改变其功能的小分子，然后开发用于结构活性关系（SAR）研究的类似物，以鉴定调节该途径的特定化合物，以及（2）在二级测定中利用这些工具并开发我们可以翻译到脊椎动物系统以探测线粒体功能的探针，包括与线粒体疾病的联系，因为从酵母到哺乳动物，蛋白质的输入是高度保守的。这些研究将产生经验证的化学探针，用于线粒体输入的机制研究和潜在诱导/消除线粒体疾病。有缺陷的TIM 22输入途径导致遗传性疾病，肥胖-肌张力障碍综合征，其导致神经变性。鉴于我们的成功，我们有信心，许多新的化合物将被确定为有关了解脊椎动物中的线粒体组装，并可能作为工具来表征肥胖-肌张力障碍综合征的分子基础。一般来说，由线粒体组装调节的事件（如细胞凋亡）的医学重要性表明，化学遗传学方法也可能导致鉴定和开发用于受功能障碍的线粒体影响的疾病的新型治疗剂。这些新化合物的鉴定，与我们在寻找靶点方面的专业知识以及我们利用它们更充分地了解机制的能力相结合，证明了我们通过MLPCN扩大这种筛选的要求是合理的。这项研究与公共卫生有关，因为它可能导致开发退行性肌肉和神经疾病的新疗法。 公共卫生相关性：该项目将开发小分子作为探针，以研究由线粒体功能缺陷引发的神经退行性和退行性肌肉疾病的原因，使用酵母作为模型系统，因为蛋白质输入途径从酵母到哺乳动物高度保守。该蛋白质为细胞产生能量，并与广泛的疾病有关，包括癌症和退行性肌肉和神经疾病。从长远来看，该项目可能会导致在这些疾病中调节线粒体功能的治疗方法的发展。

DESCRIPTION (provided by applicant): Defects in mitochondrial biogenesis lead to a broad range of diseases including neurodegeneration, stroke, myocardial infarction, ischemia, and cancer; however, therapies to correct such diseases are not readily available. We propose to conduct a high throughput screen in the Molecular Libraries Production Center Network (MLPCN) identical to the one that, on a smaller screening scale, has already successfully identified inhibitors of the mitochondrial TIM22 protein import pathway that is required for the assembly of inner membrane proteins in the model organism Saccharomyces cerevisiae. Specifically, we have devised a growth-based assay using a yeast temperature-sensitive mutant that is compromised for growth at a high temperature because of a defective import system. In the screen, we select for small molecules that are synthetically lethal with the mutant strain at a temperature that normally permits growth. Using this assay, we have identified small molecules that specifically target the TIM22 import pathway in yeast mitochondria. The aims of this proposal are to (1) identify small molecules that target the TIM22 import pathway and alter its function and then develop analogs for structure activity relationship (SAR) studies to identify specific chemical compounds that modulate this pathway and (2) utilize these tools in secondary assays and develop probes that we can translate to vertebrate systems to probe mitochondrial function, including the link to mitochondrial diseases, because protein import is highly conserved from yeast to mammals. These studies will result in validated chemical probes for mechanistic studies of mitochondrial import and for potentially inducing/abrogating mitochondrial diseases. A defective TIM22 import pathway leads to the inherited disease, deafness-dystonia syndrome, which results in neurodegeneration. Given our success, we are confident that many novel compounds will be identified that are pertinent for understanding mitochondrial assembly in vertebrates and potentially serving as tools to characterize the molecular basis of deafness-dystonia syndrome. Generally, the medical importance of events regulated by mitochondrial assembly, such as apoptosis, indicates that the chemical genetic approach may also lead to the identification and development of novel therapeutic agents for diseases affected by dysfunctional mitochondria. Identification of these novel compounds, tied with our expertise in finding targets and our ability to exploit them to more fully understand mechanism, justifies our request to expand this screen through the MLPCN. This study is relevant to public health because it may lead to the development of new therapeutics for degenerative muscular and neural diseases. PUBLIC HEALTH RELEVANCE: This project will develop small molecules as probes to investigate the cause of neurodegenerative and degenerative muscular diseases that are initiated by defects in mitochondrial function, using yeast as a model system because protein import pathways are highly conserved from yeast to mammals. The mitochondrion generates energy for the cell and is linked to a broad range of diseases, including cancer and degenerative muscular and neural diseases. Long-term, this project may lead to the development of therapeutics that modulates mitochondrial function in these diseases.