High throughput screening assays to identify small molecules that target the ClpX

通过高通量筛选分析来识别靶向 ClpX 的小分子

• 批准号: 7994954
• 负责人: CAROLYN K SUZUKI
• 金额: $ 17.05万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7994954
• 关键词: 26S proteasome; ATP Hydrolysis; ATP phosphohydrolase; ATP-Dependent Proteases; Affinity; Age; Aging; Antineoplastic Agents; B-Cell Lymphomas; Biochemical; Biological Assay; Bortezomib; Cardiomyopathies; Catalytic Domain; Cell Cycle Progression; Cell Differentiation process; Cell Line; Cell Nucleus; Cell physiology; Cells; Cessation of life; Chemotherapy-Oncologic Procedure; Cleaved cell; Clinical; Cultured Cells; Cytosol; Data; Development; Disease; Drug Delivery Systems; Endoplasmic Reticulum; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Event; Family; Figs - dietary; Fluorescence Polarization; Fluorometry; Goals; Hydrolysis; Immune response; Light; Link; Lymphoma; Malignant Neoplasms; Measures; Mediating; Membrane; Mitochondria; Mitochondrial DNA; Mitochondrial Matrix; Mitochondrial Proteins; Molecular; Multiple Myeloma; Myocardial dysfunction; Nerve Degeneration; Neurodegenerative Disorders; Neuromuscular Diseases; Noise; Oxidative Phosphorylation; Peptide Hydrolases; Peptides; Phase I Clinical Trials; Physiological; Process; Proteasome Inhibitor; Proteins; Proteolysis; Protocols documentation; Quality Control; Reading; Reporter; Respiration; Role; Screening procedure; Series; Signal Transduction; Solid Neoplasm; Specificity; Stress; System; Testing; Therapeutic; Time; assay development; base; biological adaptation to stress; cancer therapy; clinical application; drug discovery; endopeptidase La; endoplasmic reticulum stress; environmental adaptation; high throughput screening; inhibitor/antagonist; leukemia; mitochondrial dysfunction; multicatalytic endopeptidase complex; new therapeutic target; novel; programs; protein aggregation; public health relevance; response; small molecule; stable cell line; success; tool; treatment strategy; unpublished works

DESCRIPTION (provided by applicant): ATP-dependent roteases are a unique family of proteolytic enzymes that require energy to unfold protein substrates prior to peptide bond hydrolysis. In the cytosol and nucleus, the 26S proteasome is the only ATP-dependent protease, which functions to eliminate abnormal and damaged proteins, and to regulate cell differentiation, cell cycle progression, and the immune response. The proteasome has emerged as an important drug target in the treatment of multiple myeloma, and possibly other cancers. Within mitochondria, there are two soluble ATP- dependent proteases in the matrix-ClpXP and Lon. Our unpublished work suggests that these mitochondrial proteases are novel targets for drug discovery, and that inhibitors of these enzymes, either alone or in combination with proteasome inhibitors, may provide new and targeted therapeutic strategies for the treatment of cancer. In addition, activators of mitochondrial ATP-dependent proteolysis may have therapeutic potential in the treatment of neurodegenerative and/or myocardial dysfunctions that are linked to mitochondrial protein aggregation. The goal of this project is to develop high throughput screening (HTS) assays to identify small molecule inhibitors and activators of ClpXP, and to distinguish small molecules that target ClpXP versus Lon. These two ATP-dependent proteases have fundamentally distinct roles in the cellular response to stress and environmental adaptation, and thus represent distinct drug targets. For example, ClpXP expression is induced by the accumulation of unfolded proteins within mitochondria, whereas Lon expression is unchanged. However, Lon but not ClpXP expression is upregulated by unfolded proteins within the endoplasmic reticulum (ER), and is likely important in the ER stress response, which is important in numerous diseases states and aging. As ClpXP and Lon have fundamentally different cellular functions, it is essential to identify compounds that specifically target these enzymes, if we are to understand and exploit their therapeutic potential. ClpXP and Lon proteases are soluble and readily purified with high yield, making them suitable for high throughput biochemical assays. We have already made substantial progress in developing high throughput screening assays for the Lon protease. Thus, this project aims at developing a rigorous set of high throughput screening assays optimized for the ClpXP protease. (1) We will develop and optimize a primary screen that specifically measures the protease activity of purified ClpXP. We will also optimize secondary- and counter-screening assays for characterizing and prioritizing active compounds that target ClpXP. Secondary screens will determine the effect of active compounds on the ATPase and peptidase activity of ClpXP; and counter screens will determine the specificity of active compounds by measuring their effects on Lon and/or the proteasome. (2) In addition, we will develop a high-content screening assay for ClpXP-dependent proteolysis using a ClpXP reporter substrate. Using this cell-based assay we will determine whether compounds identified in the primary screen are membrane-permeable, and whether under physiological conditions they activate or inhibit ClpXP-mediated degradation of a mitochondrial reporter substrate. Taken together, the primary, secondary, and counter screens, in combination with a cell-based assay will permit us to identify, prioritize and validate active compounds that target the mitochondrial ClpXP protease. PUBLIC HEALTH RELEVANCE: The aim of this one-year R21 project is to develop a high-throughput screening (HTS) program for identifying small molecules, which specifically and potently inhibit or activate the ClpXP ATP- dependent protease. A rigorous set of biochemical and cell-based assays that are amenable to HTS, will measure the protease and ATPase activities of ClpXP. These assays will be used to discriminate small molecules that target the ClpXP protease from the Lon protease, which is the only other ATP-dependent protease within the mitochondrial matrix. Identifying small molecule inhibitors or activators will be the first step in developing compounds that permit us to discrimate the physiological functions of ClpXP and Lon, and will assist in defining ClpXP and Lon as potential clinical targets in mitochondrial dysfunction, which is associated with numerous neuromuscular disorders, cardiomyopathies, cancers and aging. Inhibitors of ClpXP and/or Lon have likely application as anti-cancer agents used either alone or in combination with other chemotherapeutic strategies. This possibility is supported by preliminary data demonstrating that ClpXP and Lon are up-regulated in a variety of lymphoma cell lines, and that compounds used to treat leukemia and solid tumors also inhibit Lon-mediated proteolysis. In addition, activators of ClpXP and/or Lon may also be exploited in the treatment of neurodegenerative disorders linked to mitochondrial protein aggregation.

描述（由申请人提供）：ATP依赖性蛋白酶是一个独特的蛋白水解酶家族，在肽键水解之前需要能量来展开蛋白质底物。在细胞质和细胞核中，26S 蛋白酶体是唯一的 ATP 依赖性蛋白酶，其功能是消除异常和受损的蛋白质，并调节细胞分化、细胞周期进程和免疫反应。蛋白酶体已成为治疗多发性骨髓瘤和其他癌症的重要药物靶点。在线粒体内，基质中有两种可溶性 ATP 依赖性蛋白酶 - ClpXP 和 Lon。我们未发表的工作表明，这些线粒体蛋白酶是药物发现的新靶点，并且这些酶的抑制剂，无论是单独使用还是与蛋白酶体抑制剂组合，都可以为癌症的治疗提供新的靶向治疗策略。此外，线粒体 ATP 依赖性蛋白水解激活剂可能具有治疗与线粒体蛋白聚集相关的神经退行性和/或心肌功能障碍的潜力。该项目的目标是开发高通量筛选 (HTS) 检测方法，以鉴定 ClpXP 的小分子抑制剂和激活剂，并区分针对 ClpXP 和 Lon 的小分子。这两种 ATP 依赖性蛋白酶在细胞对应激和环境适应的反应中具有根本不同的作用，因此代表不同的药物靶点。例如，ClpXP 表达是由线粒体内未折叠蛋白的积累诱导的，而 Lon 表达则没有变化。然而，内质网 (ER) 内的未折叠蛋白上调 Lon 表达，但不上调 ClpXP 表达，并且可能在 ER 应激反应中发挥重要作用，而 ER 应激反应在许多疾病状态和衰老中很重要。由于 ClpXP 和 Lon 具有根本不同的细胞功能，因此如果我们要了解和利用它们的治疗潜力，就必须鉴定专门针对这些酶的化合物。 ClpXP 和 Lon 蛋白酶是可溶的，易于纯化且产量高，使其适合高通量生化测定。我们在开发 Lon 蛋白酶的高通量筛选方法方面已经取得了实质性进展。因此，该项目旨在开发一套针对 ClpXP 蛋白酶优化的严格高通量筛选测定方法。 (1) 我们将开发和优化专门测量纯化 ClpXP 蛋白酶活性的初级筛选。我们还将优化二次和反筛选测定，以表征和优先考虑针对 ClpXP 的活性化合物。二次筛选将确定活性化合物对 ClpXP ATP 酶和肽酶活性的影响；计数器筛选将通过测量活性化合物对 Lon 和/或蛋白酶体的影响来确定活性化合物的特异性。 (2) 此外，我们将开发一种使用 ClpXP 报告底物进行 ClpXP 依赖性蛋白水解的高内涵筛选测定法。使用这种基于细胞的测定，我们将确定在初级筛选中鉴定的化合物是否具有膜渗透性，以及在生理条件下它们是否激活或抑制 ClpXP 介导的线粒体报告底物降解。总而言之，初级、次级和反筛选，与基于细胞的测定相结合，将使我们能够识别、优先排序和验证针对线粒体 ClpXP 蛋白酶的活性化合物。 公共健康相关性：这个为期一年的 R21 项目的目的是开发一个高通量筛选 (HTS) 程序，用于识别小分子，这些小分子特异性且有效地抑制或激活 ClpXP ATP 依赖性蛋白酶。一套严格的生化和基于细胞的检测方法适用于 HTS，将测量 ClpXP 的蛋白酶和 ATP 酶活性。这些测定将用于区分靶向 ClpXP 蛋白酶和 Lon 蛋白酶的小分子，Lon 蛋白酶是线粒体基质内唯一的其他 ATP 依赖性蛋白酶。鉴定小分子抑制剂或激活剂将是开发化合物的第一步，这些化合物使我们能够区分 ClpXP 和 Lon 的生理功能，并将有助于将 ClpXP 和 Lon 定义为线粒体功能障碍的潜在临床靶标，线粒体功能障碍与许多神经肌肉疾病、心肌病、癌症和衰老有关。 ClpXP 和/或 Lon 抑制剂可能作为抗癌剂单独使用或与其他化疗策略联合使用。初步数据支持了这种可能性，表明 ClpXP 和 Lon 在多种淋巴瘤细胞系中上调，并且用于治疗白血病和实体瘤的化合物也抑制 Lon 介导的蛋白水解作用。此外，ClpXP 和/或 Lon 激活剂也可用于治疗与线粒体蛋白聚集相关的神经退行性疾病。