M.tuberculosis Protease ClpP1P2 - An attractive drug target

结核分枝杆菌蛋白酶 ClpP1P2 - 一个有吸引力的药物靶点

• 批准号: 8182672
• 负责人: ALFRED L GOLDBERG
• 金额: $ 16.95万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8182672
• 关键词: ATP phosphohydrolase; Active Sites; Address; Affect; Antibiotics; Area; Bacteria; Biochemical; Biological Assay; Complex; Cytoplasm; Cytosol; Development; Disease; Drug Delivery Systems; Enzymes; Escherichia coli; Family; Genes; Genetic; Goals; Individual; Laboratories; Lead; Lysosomes; Mammalian Cell; Measurement; Methods; Mycobacterium smegmatis; Mycobacterium tuberculosis; Pathway interactions; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Property; Proteins; Protocols documentation; Reporting; Resistance; Screening procedure; Staging; System; Tuberculosis; Ubiquitin; United States National Institutes of Health; Work; base; drug development; experience; high throughput screening; improved; inhibitor/antagonist; member; multicatalytic endopeptidase complex; novel; preference; programs; protein degradation; small molecule; small molecule libraries; success

DESCRIPTION (provided by applicant): Tuberculosis is a devastating disease affecting 100 million individuals around the world, and the causative agent Mycobacterium Tuberculosis (Mtb) has become increasingly resistant to the available antibiotics. Consequently, it is vital to identify Mtb enzymes that are essential for its viability and to find drugs that selectively inhibit their functions. In collaborative studies, we have established by genetic approaches that clpP1 and clpP2 genes, which appear to encode intracellular proteases, are essential for the viability and infectivity of Mtb. Our laboratory has recently shown that Mtb ClpP1 and ClpP2 proteins are components of a single proteolytic complex, and we have developed methods to express, isolate and assay the active enzyme, which has a number of novel structural and regulatory properties. In E. Coli, the homologous enzyme, ClpP, is a well- characterized protease complex that is composed of 14 identical subunits and functions in protein degradation as a part of the ATP-dependent proteolytic complexes. Mtb ClpP1P2, however, is a 14-mer composed of a ClpP1 and a ClpP2 heptameric ring. In addition, we identified several small molecule (peptides or peptide derivatives) activators that are required for the formation of the active mixed ClpP1P2 complex and for its activity (even in the absence of a regulatory ATPase). We identified a number of fluorescent peptide substrates that can be used in a sensitive, reliable assay and showed that active sites on the ClpP1 and ClpP2 rings differ in substrate preference. Since ClpP is not present in the cytoplasm of mammalian cells, where protein breakdown involves very different enzymes (by the ubiquitin-proteasome pathway or in lysosomes), specific inhibitors of ClpP or of its novel activation mechanism are highly attractive drug targets. Our major goals will be to further characterize Mtb ClpP1P2 and its properties and to adapt our enzymatic assay to a high throughput format that will allow us to screen for small molecule inhibitors and activators. Because a class of antibiotics has been reported that causes uncontrolled activation of the ClpP protease in B.subtilis with toxic consequences, we shall also screen for agents that activate ClpP. These inhibitors or activators could then serve as lead compounds in a drug development program. PUBLIC HEALTH RELEVANCE: Tuberculosis is a devastating disease affecting 100 million individuals around the world. This bacterium has become increasingly resistant to the available antibiotics. Our collaborator Dr. Eric Rubin and co-workers have found that genes (clpP1 and clpP2) encoding protease ClpP are essential for the viability and infectivity of Mycobacterium Tuberculosis (Mtb). Therefore this enzyme is a highly attractive drug target, especially since it does not exist in the cytosol of mammalian cells. Our laboratory succeeded in isolating and characterizing this member of ClpP family, which has many novel biochemical properties. Our major goals will be to optimize a high throughput assay of this enzyme that will allow screen for small molecule inhibitors (or activators) that might serve as lead compounds in a drug development program.

描述（由申请人提供）：结核病是一种毁灭性的疾病，影响着全世界 1 亿人，其病原体结核分枝杆菌 (Mtb) 对现有抗生素的耐药性越来越强。因此，识别对其生存至关重要的 Mtb 酶并找到选择性抑制其功能的药物至关重要。在合作研究中，我们通过遗传方法确定了 clpP1 和 clpP2 基因（它们似乎编码细胞内蛋白酶）对于 Mtb 的生存力和感染性至关重要。我们的实验室最近表明，结核分枝杆菌 ClpP1 和 ClpP2 蛋白是单一蛋白水解复合物的组成部分，并且我们开发了表达、分离和测定活性酶的方法，该酶具有许多新颖的结构和调节特性。在大肠杆菌中，同源酶 ClpP 是一种特征明确的蛋白酶复合物，由 14 个相同的亚基组成，作为 ATP 依赖性蛋白水解复合物的一部分在蛋白质降解中发挥作用。然而，Mtb ClpP1P2 是由 ClpP1 和 ClpP2 七聚环组成的 14 聚体。此外，我们还鉴定了几种小分子（肽或肽衍生物）激活剂，它们是活性混合 ClpP1P2 复合物的形成及其活性（即使在没有调节性 ATP 酶的情况下）所必需的。我们鉴定了许多可用于灵敏、可靠测定的荧光肽底物，并表明 ClpP1 和 ClpP2 环上的活性位点在底物偏好方面有所不同。由于 ClpP 不存在于哺乳动物细胞的细胞质中，其中蛋白质分解涉及非常不同的酶（通过泛素蛋白酶体途径或在溶酶体中），因此 ClpP 或其新颖激活机制的特异性抑制剂是非常有吸引力的药物靶点。我们的主要目标是进一步表征 Mtb ClpP1P2 及其特性，并使我们的酶测定适应高通量格式，从而使我们能够筛选小分子抑制剂和激活剂。由于已报道一类抗生素会导致枯草芽孢杆菌中 ClpP 蛋白酶不受控制的激活，并产生毒性后果，因此我们还应筛选激活 ClpP 的药物。这些抑制剂或激活剂可以作为药物开发计划中的先导化合物。 公共卫生相关性：结核病是一种毁灭性的疾病，影响着全世界一亿人。这种细菌对现有抗生素的耐药性越来越强。我们的合作者 Eric Rubin 博士及其同事发现编码蛋白酶 ClpP 的基因（clpP1 和 clpP2）对于结核分枝杆菌 (Mtb) 的生存力和感染性至关重要。因此，这种酶是一个非常有吸引力的药物靶点，特别是因为它不存在于哺乳动物细胞的细胞质中。我们的实验室成功分离并表征了 ClpP 家族的这一成员，它具有许多新颖的生化特性。我们的主要目标是优化这种酶的高通量测定，从而筛选可能作为药物开发计划中的先导化合物的小分子抑制剂（或激活剂）。