The ClpP protease as a therapeutic target in bacterial and mammalian cells

ClpP 蛋白酶作为细菌和哺乳动物细胞的治疗靶点

• 批准号: 8938126
• 负责人: MICHAEL MAURIZI
• 金额: $ 26.03万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8938126
• 关键词: ATP phosphohydrolase; Active Sites; Adverse effects; Affect; Affinity; Amino Acids; Antibiotic Resistance; Antibiotics; Apical; Award; Bacteria; Binding; Biochemical; Biological; Biological Assay; Biological Process; Biological Products; Cancer cell line; Cells; Chemical Structure; Chemicals; Collaborations; Community Hospitals; Complement; Complex; Data; Depsipeptides; Development; Discrimination; Docking; Drug Targeting; Effectiveness; Elements; Enterococcus; Environment; Escherichia; Escherichia coli; Eukaryota; Funding; Genetic Screening; Genomics; Goals; Grant; Growth; Health Care Costs; Heating; Homeostasis; Hospitals; Human; Hydrogen; Hydrophobic Interactions; Infection; Klebsiella; Laboratories; Lactones; Lead; Length of Stay; Life; Literature; Mammalian Cell; Mammals; Minor; Mitochondria; Modification; Multi-Drug Resistance; Mutate; Mutation; Osmotic Shocks; Pathway interactions; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Play; Procedures; Process; Property; Proteins; Research; Resistance; Role; Scientist; Site; Solutions; Species Specificity; Specificity; Starvation; Streptococcus; Streptococcus pneumoniae; Structure; Surface; Synthesis Chemistry; Testing; Time; United States; United States National Institutes of Health; Variant; Vertebral column; Work; X-Ray Crystallography; adduct; antimicrobial; antimicrobial drug; base; cell killing; comparative; compound 18; design; endopeptidase Clp; flexibility; genetic regulatory protein; high throughput screening; in vitro activity; inhibitor/antagonist; killings; methicillin resistant Staphylococcus aureus; microorganism; mutant; novel; protein degradation; resistant strain; response; screening; small molecule libraries; therapeutic target; tool; unfoldase

This project has two main elements. The major effort began two years ago and involved collaboration with scientists at the NIH Chemical Genomics Center (NCGC) to conduct a high-throughput screen (HTS) of a large chemical library to search for compounds that activate ClpP peptidase and protease activity in a manner similar to the ADEP antibiotics. This project was partially funded through an R03 award (1 R03 MH095569) granted to me in 2012. The interactions between ADEP and ClpP, as shown by X-ray crystallography, suggest that there should be a high likelihood of finding organic molecules that display a rigid structure that mimics the aromatic/aliphatic part of ADEP, dock to ClpP, and exert allosteric effects on its activity. The primary contacts between ADEP and ClpP involve hydrophobic interactions between an aromatic ring in ADEP and a deep pocket on the apical surface of ClpP. In addition, there are hydrophobic interactions between an aliphatic chain in ADEP and a hydrophobic groove that extends from the hydrophobic pocket toward the axial channel of ClpP. Other minor interactions include hydrogen binding involving backbone atoms from a short peptide segment of ADEP. The depsipeptide portion of ADEP has very little interaction with ClpP and serves primarily to restrict the conformational flexibility of the aliphatic regions in ADEP, which are fixed in a configuration that locks into the docking site. The solution structure of ADEP alone confirms that there is little induced change in its upon binding to ClpP. After a large scale screening of over 300,000 compounds, about 18 compounds were identified as potential inhibitors of ClpP and about 30 were identified as potential activators. The compounds are now being tested in more detail for their effects on various activities of ClpP. Compounds that that are identified as validated activators of inhibitors will be provided in larger quantities for further studies and for structural studies to identify the sites and mode of binding. They will be assayed further in my laboratory to obtain a more complete profile of binding affinity, activating effect on both peptide and protein substrates, and comparative specificity for human, E. coli, and B. subtilis ClpPs. Compounds will then be tested for antimicrobial activity against laboratory strains of E. coli and B. subtilis. Compounds will also be tested for their growth inhibitory activity against several human cancer cell lines. Once promising lead compounds have been identified and screened by the various secondary assays mentioned, the synthetic chemistry team at NCGC will begin designing synthetic strategies for making the compounds and variations of the compounds to develop new versions that are optimized for binding to ClpP and for effectiveness against cultures of bacteria. To complement the efforts to identify new compounds that mimic ADEPs in their binding to ClpP, we conducted a genetic screen to obtain mutants of ClpP that have altered binding properties and possibly altered allosteric responses to binding of ADEP. ADEPs bind to the docking site on the apical surface of ClpP used by ClpX and ClpA/C in forming the biologically functional ClpXP and ClpAP complexes. We developed a sensitive selection procedure that identified mutants of ClpP that were resistant to ADEP but retained enzymatic activity with ClpX. The selection was based on the ability of ClpXP to degrade proteins with an 11-amino acid degradation tag (called an SsrA tag) at the C-terminus. From a group of multiply mutated ClpPs we have isolated six forms of ClpP bearing single mutations. Cells expressing the mutants retain activity in degrading the SsrA-tagged protein and are resistant to ADEP to varying degrees. We have purified the mutant proteins are in the process of studying their biochemical and enzymatic activities in vitro. The goal of this work is to identify the critical residues in ClpP that are involved in both binding of ADEPs and ClpX and in the allosteric response that communicates to the axial channel and causes the channel to be expended and allow indiscriminate protein entry. Mutated forms of ClpP that respond differently to ADEP and ClpX could show different binding affinity or binding rates or could be affected in residues that make new interactions that stabilize the activated structure of ClpP. In a related effort, we have initiated an effort to synthesize beta-lactone inhibitors of ClpP. Initially we are making two inhibitors that have been described in the literature, and plans are to make modifications to the procedure to introduce other substituents that should contribute additional binding affinity to ClpP. These inhibitors will be reacted with purified ClpP to study the effects on the quaternary structure and to obtain crystal structure data to elucidate how they are bound in the ClpP active site.

该项目有两个主要要素。主要的努力始于两年前，并涉及与NIH化学基因组学中心（NCGC）的科学家合作，以进行大型化学文库的高通量屏幕（HTS），以搜索以类似于EDEP抗生素类似的方式激活CLPP肽酶和蛋白酶活性的化合物。该项目是通过R03奖（1 R03 MH095569）资助的。如X射线晶体学所示，ADEP和CLPP之间的相互作用表明，应该很可能会发现有机分子，这些有机分子可能会发现刚性结构，这些结构模仿了芳香/aliphatic expect of Adpp的clpep and clpp and clpec and doc cl cl expe and clp exp，又有很可能。 ADEP和CLPP之间的主要接触涉及EDEP中的芳香环与CLPP顶部的深口袋之间的疏水相互作用。另外，EDEP中的脂肪族链与疏水性凹槽之间存在疏水相互作用，该疏水性凹槽从疏水口袋延伸到Clpp的轴向通道。其他次要相互作用包括涉及来自EDEP短肽段的骨架原子的氢结合。 ADEP的深度肽部分与CLPP几乎没有相互作用，主要用于限制ADEP中脂肪族区域的构象灵活性，而ADEP中的脂肪族柔韧性则固定在锁定到对接位点的配置中。单独的ADEP的溶液结构证实，与CLPP结合后，其诱导的变化很小。在大规模筛选300,000多种化合物后，将约18种化合物确定为CLPP的潜在抑制剂，约30种被确定为潜在激活剂。现在，对化合物对CLPP的各种活动的影响进行了更详细的测试。将大量研究和结构研究以识别位点和结合方式，以更大的量提供被鉴定为抑制剂验证活化剂的化合物。它们将在我的实验室中进一步分析，以获得更完整的结合亲和力，激活对肽和蛋白质底物的激活作用以及对人，大肠杆菌和枯草芽孢杆菌CLPP的比较特异性。然后，将测试化合物对大肠杆菌和枯草芽孢杆菌的实验室菌株的抗菌活性。还将测试化合物对几种人类癌细胞系的生长抑制活性。一旦通过提到的各种次要测定鉴定并筛选了有希望的铅化合物，NCGC的合成化学团队将开始设计合成策略，以制造化合物的化合物和变化，以开发新版本的新版本，以与CLPP结合并针对CLPP结合并针对细菌的有效性结合。为了补充识别模仿其与CLPP结合的新化合物的努力，我们进行了遗传筛选，以获得具有改变结合特性的CLPP突变体，并可能改变了对ADEP结合的变构响应。 ADEP与Clpx和Clpa/C使用的CLPP的顶部表面上的对接位点结合，以形成生物功能性的CLPXP和CLPAP复合物。我们开发了一种敏感的选择程序，该程序鉴定出对EDEP抗性但使用CLPX保留酶促活性的CLPP突变体。该选择是基于CLPXP在C末端降解蛋白质（称为SSRA标签）的蛋白质的能力。从一组乘突变的CLPP中，我们分离了六种形式的CLPP，具有单个突变。表达突变体的细胞在降解SSRA标记的蛋白质中保留活性，并且在不同程度上对ADEP具有抗性。我们已经纯化了突变蛋白在体外研究其生化和酶促活性的过程。这项工作的目的是确定CLPP中的关键残基，这些残基均参与EDEPS和CLPX的结合以及在轴向通道通信的变构响应中，并导致该通道被消耗并允许不加选择的蛋白质进入。对ADEP和CLPX的反应不同的CLPP的突变形式可能显示出不同的结合亲和力或结合速率，或者可能会影响使新相互作用稳定CLPP激活结构的残基。在一项相关的工作中，我们开始努力合成CLPP的β-内乳转酮抑制剂。最初，我们正在制作文献中描述的两个抑制剂，并计划对程序进行修改，以引入其他化取代基，这些取代基应与CLPP产生额外的结合亲和力。这些抑制剂将与纯化的CLPP反应，以研究对第四纪结构的影响并获得晶体结构数据，以阐明它们在CLPP活性位点的结合。