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

基本信息

批准号：
9343932
负责人：
MICHAEL MAURIZI
金额：
$ 15.95万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/9343932
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 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 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 targeted agent therapeutic target tool unfoldase

项目摘要

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。这项主要工作始于两年前，涉及与 NIH 化学基因组中心 (NCGC) 的科学家合作，对大型化学库进行高通量筛选 (HTS)，以寻找以某种方式激活 ClpP 肽酶和蛋白酶活性的化合物。与 ADEP 抗生素类似。该项目的部分资金来自 2012 年授予我的 R03 奖（1 R03 MH095569）。X 射线晶体学显示的 ADEP 和 ClpP 之间的相互作用表明，应该很有可能找到显示出模拟 ADEP 芳香族/脂肪族部分的刚性结构，对接 ClpP，并对其活性发挥变构作用。 ADEP 和 ClpP 之间的主要接触涉及 ADEP 中的芳环和 ClpP 顶表面上的深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。其他次要的相互作用包括涉及来自 ADEP 短肽片段的主链原子的氢结合。 ADEP 的缩酚肽部分与 ClpP 的相互作用非常少，主要用于限制 ADEP 中脂肪族区域的构象灵活性，这些区域固定在锁定对接位点的构型中。 ADEP 的溶液结构单独证实了其与 ClpP 结合后几乎没有诱导变化。经过对超过 300,000 种化合物的大规模筛选，约 18 种化合物被鉴定为潜在的 ClpP 抑制剂，约 30 种化合物被鉴定为潜在的激活剂。目前正在更详细地测试这些化合物对 ClpP 各种活性的影响。被鉴定为经过验证的抑制剂激活剂的化合物将大量提供用于进一步研究和结构研究，以确定结合位点和模式。它们将在我的实验室进行进一步分析，以获得更完整的结合亲和力、对肽和蛋白质底物的激活作用以及对人类、大肠杆菌和枯草芽孢杆菌 ClpPs 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二次分析鉴定和筛选了有前途的先导化合物，NCGC 的合成化学团队将开始设计用于制造化合物和化合物变体的合成策略，以开发针对 ClpP 结合和有效性进行优化的新版本对抗细菌培养物。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 ClpP 突变体，这些突变体改变了结合特性，并可能改变了对 ADEP 结合的变构反应。 ADEP 与 ClpX 和 ClpA/C 使用的 ClpP 顶端表面的对接位点结合，形成具有生物功能的 ClpXP 和 ClpAP 复合物。我们开发了一种灵敏的选择程序，可鉴定对 ADEP 具有抗性但保留 ClpX 酶活性的 ClpP 突变体。该选择基于 ClpXP 降解 C 末端带有 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，我们分离出了六种带有单突变的 ClpP 形式。表达突变体的细胞保留了降解 SsrA 标记蛋白的活性，并且对 ADEP 具有不同程度的抗性。我们已经纯化了突变蛋白，正在研究它们的体外生化和酶活性。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，以及与轴向通道通讯并导致通道扩张并允许任意蛋白质进入的变构反应。对 ADEP 和 ClpX 反应不同的 ClpP 突变形式可能会表现出不同的结合亲和力或结合率，或者可能会受到残基的影响，从而产生新的相互作用，从而稳定 ClpP 的活化结构。在一项相关的工作中，我们已经开始合成 ClpP 的 β-内酯抑制剂。最初，我们正在制造文献中描述的两种抑制剂，并计划对程序进行修改，以引入其他取代基，这些取代基应为 ClpP 提供额外的结合亲和力。这些抑制剂将与纯化的 ClpP 反应，研究对四级结构的影响并获得晶体结构数据，以阐明它们如何与 ClpP 活性位点结合。