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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至关重要对环境压力的反应。这些酶是 ATP 驱动的蛋白水解机器，选择性降解异常蛋白质并调节代谢过程。在许多疾病状态下和衰老，线粒体内活性氧的产生增加导致蛋白质氧化和聚集。作为对策，线粒体 Lon 蛋白酶例如，降解氧化乌头酸酶，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与衰老直接相关，心脏病和神经肌肉疾病；而蛋白质聚集对于许多人来说很常见神经退行性疾病。此外，最近的研究表明 Lon 介导的蛋白水解作用可能在肿瘤发生和瘤内细胞对缺氧的适应中发挥重要作用。很遗憾，没有特定的高亲和力线粒体 ATP 依赖性抑制剂或激活剂蛋白酶。该提案的目标是发展用于识别和验证选择性激活或抑制 Lon 的化合物的测定蛋白酶。目标 1 是开发和优化用于测量 Lonmedia 的初级筛选方法使用时间分辨荧光法降解报告肽底物。可行性及这些测定的重现性将使用市售化学品来证明图书馆。目标 2 是开发和优化二次和反筛选测定法，这将提供有关化合物活性机制的信息，并区分以下化合物：与针对其他线粒体或非线粒体蛋白酶的那些具有 Lon 特异性。小的线粒体 ATP 依赖性蛋白水解的分子激活剂在治疗与线粒体蛋白相关的神经退行性和/或心肌功能障碍聚合。我们的初步数据表明线粒体 ATP 依赖性抑制剂蛋白水解可作为抗癌剂，单独或具有潜在的临床应用与其他化疗策略相结合。综合来看，Lon 和其他线粒体 ATP 依赖性蛋白酶可能是新的、可行的药物靶标。