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，影响了世界各地的1亿个人，而致病剂结核分枝杆菌（MTB）已经越来越对可用抗生素具有抗药性。因此，至关重要的是，鉴定MTB酶对其生存能力至关重要，并找到有选择地抑制其功能的药物。在协作研究中，我们通过遗传方法确定了Clpp1和Clpp2基因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，MTB CLPP1和CLPP2蛋白是单个蛋白水解复合物的组成部分，并且我们开发了表达，分离和分析活性酶的方法，该酶具有许多新型的结构和调节性能。在大肠杆菌中，同源酶CLPP是一种特征良好的蛋白酶复合物，由14个相同的亚基组成，蛋白质降解中的功能是ATP依赖性蛋白水解复合物的一部分。但是，MTB CLPP1P2是由CLPP1和CLPP2七聚体环组成的14-MER。此外，我们确定了几个小分子（肽或肽衍生物）激活剂，它们是形成活性混合Clpp1p2复合物及其活性所必需的（即使在没有调节ATPase的情况下）。我们鉴定了许多可用于敏感，可靠的测定法中的荧光肽底物，并表明Clpp1和Clpp2环上的活性位点在底物偏好方面有所不同。由于哺乳动物细胞的细胞质中不存在CLPP，因此蛋白质分解涉及非常不同的酶（通过泛素 - 蛋白酶体途径或溶酶体中），因此CLPP的特定抑制剂或其新型激活机制是非常有吸引力的药物目标。我们的主要目标是进一步表征MTB CLPP1P2及其特性，并将酶促测定适应高通量格式，这将使我们能够筛选出小分子抑制剂和激活剂。由于已经报道了一类抗生素，该抗生素会导致B.subtilis中CLPP蛋白酶的激活不受控制，因此我们还将筛选激活CLPP的药物。然后，这些抑制剂或激活剂可以作为药物开发计划中的铅化合物。 公共卫生相关性：结核病是一种毁灭性疾病，影响了世界各地1亿人。该细菌对可用的抗生素越来越抗性。我们的合作者埃里克·鲁宾（Eric Rubin）和同事发现，编码蛋白酶CLPP的基因（CLPP1和CLPP2）对于结核分枝杆菌（MTB）的生存能力和感染性至关重要。因此，该酶是一个极具吸引力的药物靶标，尤其是因为它不存在于哺乳动物细胞的细胞质中。我们的实验室成功地隔离了CLPP家族成员，该成员具有许多新颖的生化特性。我们的主要目标是优化该酶的高吞吐量测定法，该酶将允许筛选小分子抑制剂（或激活剂），这些酶可能用作药物开发计划中的铅化合物。