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

描述(由申请人提供)：ATP依赖的旋转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。在胞浆和胞核中，26S蛋白酶体是唯一的依赖于ATP的蛋白水解酶，其功能是清除异常和受损的蛋白质，调节细胞分化、细胞周期进程和免疫反应。蛋白酶体已经成为治疗多发性骨髓瘤的重要药物靶点，可能还有其他癌症。在线粒体内，基质中有两种可溶的ATP依赖的蛋白水解酶-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，这些酶的抑制剂，无论是单独使用还是与蛋白酶体抑制剂联合使用，都可能为癌症的治疗提供新的靶向治疗策略。此外，线粒体ATP依赖的蛋白分解激活剂可能在治疗与线粒体蛋白聚集有关的神经退行性和/或心肌功能障碍方面具有治疗潜力。该项目的目标是开发高通量筛选(HTS)方法来识别ClpXP的小分子抑制物和激活剂，并区分靶向ClpXP和Lon的小分子。这两种依赖于三磷酸腺苷的蛋白水解酶在细胞对压力和环境适应的反应中具有根本不同的作用，因此代表着不同的药物靶点。例如，ClpXP的表达是由线粒体内未折叠蛋白的积累诱导的，而Lon的表达没有变化。然而，Lon而不是ClpXP的表达是由内质网(ER)中的未折叠蛋白上调的，并且可能在内质网应激反应中起重要作用，这在许多疾病状态和衰老中都是重要的。由于ClpXP和Lon具有根本不同的细胞功能，如果我们要了解和开发它们的治疗潜力，识别专门针对这些酶的化合物是至关重要的。ClpXP和Lon蛋白水解酶具有易溶、易纯化、产率高等特点，适合于高通量生化分析。我们已经在开发高通量的Lon蛋白酶筛选方法方面取得了实质性的进展。因此，本项目旨在开发一套针对ClpXP蛋白酶进行优化的严格的高通量筛选方法。(1)我们将开发和优化一种专门测定纯化的ClpXP蛋白酶活性的初步筛选方法。我们还将优化二次和反筛选试验，以确定针对ClpXP的活性化合物的特征和优先顺序。二级筛选将确定活性化合物对ClpXP的ATPase和多肽酶活性的影响；而反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，我们还将建立一种使用ClpXP报告底物的ClpXP依赖蛋白水解酶的高含量筛选方法。使用这种基于细胞的分析方法，我们将确定在初级筛选中鉴定的化合物是否是膜通透性的，以及在生理条件下，它们是激活还是抑制ClpXP介导的线粒体报告底物的降解。综合起来，一次、二次和计数器筛选，结合基于细胞的分析，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。 与公众健康相关：这个为期一年的R21项目的目标是开发一种高通量筛选(HTS)计划，用于识别特定和有效地抑制或激活ClpXP ATP依赖的蛋白酶的小分子。一套严格的符合HTS的生化和基于细胞的分析方法将测量ClpXP的蛋白酶和ATPase活性。这些检测将被用来区分靶向ClpXP酶的小分子和Lon酶，Lon酶是线粒体基质中唯一的另一种依赖于ATP的蛋白酶。识别小分子抑制剂或激活剂将是开发化合物的第一步，这些化合物允许我们描述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点，线粒体功能障碍与许多神经肌肉疾病、心肌疾病、癌症和衰老有关。ClpXP和/或LON的抑制剂可能作为抗癌药物单独使用或与其他化疗策略联合使用。初步数据表明，ClpXP和Lon在各种淋巴瘤细胞系中表达上调，用于治疗白血病和实体瘤的化合物也抑制Lon介导的蛋白质分解，从而支持了这种可能性。此外，ClpXP和/或Lon的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。

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 Our unpublished work suggests that these mitochondrial proteases are novel targets for drug discovery, and that inhibitors of these enzymes, either alone or in combination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.