High throughput screens for modulators of mitochondrial ATP-dependent proteolysis

高通量筛选线粒体 ATP 依赖性蛋白水解调节剂

基本信息

批准号：
7914479
负责人：
CAROLYN K SUZUKI
金额：
$ 35.99万
依托单位：
UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-15 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7914479
关键词：
26S proteasome ATP phosphohydrolase ATP-Dependent Proteases Aconitate Hydratase Adverse effects Affinity Age Aging Antineoplastic Agents Apoptosis Biological Assay Bortezomib Cardiomyopathies Caseins Cells Data Development Disease Drug Delivery Systems Enzymes Europium FDA approved Fluorescence Fluorescent Dyes Fluorometry Funding Generations Goals Green Fluorescent Proteins Heart Diseases Homeostasis Hypoxia In Vitro Indirect Immunofluorescence Label Life Link Lipids Malignant Neoplasms Measures Mediating Membrane Potentials Metabolism Mitochondria Mitochondrial Proteins Molecular Monitor Morphology Multiple Myeloma Myocardial dysfunction NIH Program Announcements Nerve Degeneration Neurodegenerative Disorders Neuromuscular Diseases Noise Nucleic Acids Peptide Hydrolases Peptides Phase I Clinical Trials Play Preclinical Drug Evaluation Proteasome Inhibitor Proteins Proteolysis Publishing Reactive Oxygen Species Reading Reporter Reproducibility Role Screening procedure Serine Signal Transduction Solid Neoplasm Specificity Stress Tetracycline Control Time Velcade Work assay development base cellular imaging cellular targeting chemotherapy clinical application clinically relevant endopeptidase La factor A fluorophore high throughput screening inhibitor/antagonist leukemia mitochondrial dysfunction oxidation oxidative damage prevent promoter protein aggregation research study response small molecule small molecule libraries stable cell line steroidogenic acute regulatory protein time use tool transcription factor tumorigenesis

项目摘要

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.

线粒体ATP依赖性蛋白酶对于维持细胞稳态和对环境压力的反应。这些酶是ATP驱动的蛋白水解机，有选择地降解异常蛋白质并调节代谢过程。在许多疾病状态和衰老，线粒体中活性氧的产生增加导致蛋白质氧化和聚集。作为对立的，线粒体LON蛋白酶用于例如，降解氧化的刺刺蛋白酶，从而防止其不可逆的积累和聚合。蛋白质，核酸和脂质的氧化损伤直接与衰老有关，心脏病和神经肌肉疾病；而蛋白质聚集在许多人中是常见的神经退行性疾病。此外，最近的工作表明LON介导的蛋白水解可能在肿瘤发生和肿瘤内细胞对缺氧的适应中很重要。很遗憾，没有特定的高亲和力抑制剂或线粒体ATP依赖性的激活剂蛋白酶。该提议的目标是发展识别和验证化合物的测定法，选择性激活或抑制LON 蛋白酶。 AIM 1是开发和优化用于测量LONIDED的主要筛选测定法使用时间分辨的荧光测定法降解报告肽底物。可行性和这些测定的可重复性将使用市售化学物质证明库。 AIM 2是开发和优化次要和反筛查测定法，这将提供有关复合活动机制的信息，并区分化合物来自靶向其他线粒体或非用线粒体蛋白酶的人是LON特异性的。小的线粒体ATP依赖性蛋白水解的分子活化剂在与线粒体蛋白相关的神经退行性和/或心肌功能障碍的处理聚合。我们的初步数据表明，线粒体ATP依赖性的抑制剂蛋白水解可以用作抗癌药，单独使用潜在的临床应用或结合其他化学治疗策略。两者一起，朗和其他线粒体ATP依赖性蛋白酶可能是新的且可行的药物靶标。