High throughput screens for modulators of mitochondrial ATP-dependent proteolysis

线粒体ATP依赖的蛋白水解酶对维持细胞内环境的稳定至关重要 对环境压力的反应。这些酶是由ATP驱动的蛋白水解机， 选择性地降解异常蛋白质，调节代谢过程。在许多疾病州 随着年龄的增长，线粒体内活性氧的生成增加，导致 蛋白质氧化和聚集。作为一种应对措施，线粒体Lon蛋白水解酶 例如，降解氧化的乌头酸酶，从而防止其不可逆转的积累和 聚合。蛋白质、核酸和脂质的氧化损伤与衰老直接相关， 心脏病和神经肌肉疾病；而蛋白质聚集对许多人来说是常见的 神经退行性疾病。此外，最近的研究表明，Lon介导的蛋白质分解可能 在肿瘤发生和肿瘤内细胞对低氧的适应中起重要作用。不幸的是， 没有特定的线粒体ATP依赖的高亲和力抑制物或激活剂。 蛋白酶。这项提议的目标是发展 识别和验证选择性激活或抑制Lon的化合物的方法 蛋白酶。目的1是建立和优化一种检测Lonmediated的初步筛选方法 用时间分辨荧光法降解报告多肽底物。该项目的可行性和 这些分析的重复性将使用商业上可用的化学物质进行验证。 图书馆。目标2是开发和优化二次筛查和反筛查分析，这将 提供有关化合物活性机制的信息，并区分 是Lon特异的，不同于以其他线粒体或非线粒体蛋白酶为靶点的那些。小的 线粒体ATP依赖的蛋白水解酶分子激活剂在脑损伤中的潜在应用 与线粒体蛋白相关的神经退行性和/或心肌功能障碍的治疗 聚合。我们的初步数据表明，线粒体ATP依赖的抑制物 蛋白水解物可作为抗癌药物，具有单独或潜在的临床应用 与其他化疗策略相结合。总而言之，朗和其他人 线粒体ATP依赖的蛋白水解酶可能是新的和可行的药物靶点。

Mitochondrial ATP-dependent proteases are vital for maintaining cellular homeostasis and the response to environmental stress. These enzymes are ATP-powered proteolytic machines that selectively degrade abnormal proteins and regulate metabolic processes. In many disease states and aging, the increased generation of reactive oxygen species within mitochondria results in protein oxidation and aggregation. As a counter-measure, the mitochondrial Lon protease for example, degrades oxidized aconitase thereby preventing its irreversible accumulation and aggregation. The oxidative damage of proteins, nucleic acids and lipids is directly linked to aging, heart disease and neuromuscular disorders; whereas protein aggregation is common to many neurodegenerative disorders. In addition, recent work suggests that Lon-mediated proteolysis may be important in tumorigenesis and the adaptation of intratumoral cells to hypoxia. Unfortunately, there are no specific high affinity inhibitors or activators of mitochondrial ATP-dependent proteases. The goals of this proposal are to develop assays for identifying and validating compounds that selectively activate or inhibit the Lon protease. Aim 1 is to develop and optimize a primary screening assay for measuring Lonmediated degradation of reporter peptide substrate using time-resolved fluorometry. The feasibility and reproducibility of these assays will be demonstrated using commercially available chemical libraries. Aim 2 is to develop and optimize secondary- and counter- screening assays, which will provide information about the mechanism of compound activity, and distinguish compounds that are Lon-specific from those that target other mitochondrial or non-mitochondrial proteases. Small molecule activators of mitochondrial ATP-dependent proteolysis have potential application in the treatment of neurodegenerative and/or myocardial dysfunctions linked to mitochondrial protein aggregation. Our preliminary data suggest that inhibitors of mitochondrial ATP-dependent proteolysis may function as anti-cancer agents, with potential clinical application either alone or in combination with other chemotherapeutic strategies. Taken together, Lon and other mitochondrial ATP-dependent proteases may be new and viable drug targets.