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.

该项目有两个主要内容。主要的努力始于两年前，涉及与NIH化学基因组学中心（NCGC）的科学家合作，对大型化学文库进行高通量筛选（HTS），以寻找以类似于ADEP抗生素的方式激活ClpP肽酶和蛋白酶活性的化合物。该项目的部分资金来自2012年授予我的R 03奖（1 R 03 MH 095569）。ADEP和ClpP之间的相互作用，如X射线晶体学所示，表明应该有很高的可能性找到显示刚性结构的有机分子，该刚性结构模拟ADEP的芳香族/脂肪族部分，停靠到ClpP，并对其活性产生变构效应。ADEP和ClpP之间的主要接触涉及ADEP中的芳香环和ClpP顶端表面上的深口袋之间的疏水相互作用。此外，ADEP中的脂肪链与从疏水口袋向ClpP的轴向通道延伸的疏水凹槽之间存在疏水相互作用。其他次要相互作用包括涉及来自ADEP短肽段的骨架原子的氢键合。ADEP的缩肽部分与ClpP的相互作用非常小，主要用于限制ADEP中脂肪族区域的构象灵活性，这些脂肪族区域固定在锁定对接位点的构型中。单独的ADEP的溶液结构证实，在其与ClpP结合时几乎没有诱导的变化。在对超过300，000种化合物进行大规模筛选后，约18种化合物被鉴定为ClpP的潜在抑制剂，约30种被鉴定为潜在活化剂。目前正在更详细地测试这些化合物对ClpP各种活性的影响。将大量提供被鉴定为抑制剂的经验证激活剂的化合物，用于进一步研究和结构研究，以鉴定结合位点和模式。将在我的实验室进一步分析它们，以获得更完整的结合亲和力、对肽和蛋白质底物的活化作用以及对人、E. coli和B.枯草芽孢杆菌ClpPs然后测试化合物对实验室大肠杆菌菌株的抗微生物活性。coli和B.枯草杆菌。还将测试化合物对几种人癌细胞系的生长抑制活性。一旦有希望的先导化合物已被确定，并通过上述各种二级检测筛选，NCGC的合成化学团队将开始设计合成策略，用于制造化合物和化合物的变体，以开发新的版本，优化结合ClpP和对细菌培养物的有效性。为了补充鉴定模拟ADEP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的结合特性和可能改变的对ADEP结合的变构反应的ClpP突变体。ADEP与ClpP顶端表面上的对接位点结合，ClpX和ClpA/C在形成生物功能性ClpXP和ClpAP复合物中使用该对接位点。我们开发了一个敏感的选择程序，确定突变体的ClpP，耐ADEP，但保留与ClpX的酶活性。该选择基于ClpXP降解在C末端具有11个氨基酸降解标签（称为SsrA标签）的蛋白质的能力。从一组多重突变的ClpP中，我们已经分离出六种形式的具有单突变的ClpP。表达突变体的细胞保留降解SsrA标记蛋白的活性，并在不同程度上对ADEP具有抗性。我们已经纯化了突变蛋白，并在体外研究其生化和酶活性。这项工作的目标是确定ClpP中的关键残基，这些残基参与ADEP和ClpX的结合以及与轴向通道通信并导致通道扩张并允许无差别蛋白质进入的变构反应。对ADEP和ClpX反应不同的ClpP突变形式可能显示出不同的结合亲和力或结合速率，或者可能在产生稳定ClpP活化结构的新相互作用的残基中受到影响。在相关的努力中，我们已经开始努力合成ClpP的β-内酯抑制剂。最初，我们正在制备文献中描述的两种抑制剂，并且计划对该方法进行修改以引入其他取代基，这些取代基应该有助于对ClpP的额外结合亲和力。这些抑制剂将与纯化的ClpP反应，以研究对四级结构的影响，并获得晶体结构数据，以阐明它们如何结合在ClpP活性位点。