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: 8553191
• 负责人: MICHAEL MAURIZI
• 金额: $ 22.38万
• 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/8553191
• 关键词: ATP phosphohydrolase; ATP-Dependent Proteases; Adverse effects; Affect; Affinity; Amino Acids; Antibiotic Resistance; Antibiotics; Apical; Award; Bacteria; Bacteriophages; Binding; Biochemical; Biological; Biological Assay; Biological Process; Biological Products; Cancer cell line; Cell Survival; Cells; Chemical Structure; Chemicals; Cleaved cell; Collaborations; Community Hospitals; Complement; Complex; DNA; Depsipeptides; Development; Docking; Drug Delivery Systems; Drug resistance; Effectiveness; Elements; Engineering; Enterococcus; Environment; Escherichia; Escherichia coli; Eukaryota; Evaluation; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Funding; Genetic Screening; Genomics; Goals; Gram-Negative Bacteria; Grant; Growth; Health Care Costs; Heating; Holoenzymes; Homeostasis; Hospitals; Human; Hydrogen; Hydrophobic Interactions; Immune system; Infection; Klebsiella; Laboratories; Lead; Length of Stay; Libraries; Life; Ligand Binding; Light; Mammalian Cell; Mammals; Manuscripts; Measures; Membrane; Membrane Proteins; Minor; Mitochondria; Multi-Drug Resistance; Mutate; Mutation; Osmotic Shocks; Pathway interactions; Peptide Hydrolases; Peptides; Permeability; Pharmaceutical Preparations; Plasmids; Play; Preparation; Procedures; Property; Proteins; Relative (related person); Research; Resistance; Resistance development; Role; Scientist; Screening procedure; Signal Transduction; Site; Solutions; Species Specificity; Specificity; Staphylococcus aureus; Starvation; Streptococcus; Streptococcus pneumoniae; Structure; Surface; Survivors; Synthesis Chemistry; System; Testing; Therapeutic; Time; Transplant Recipients; United States; United States National Institutes of Health; Variant; Vertebral column; Work; X-Ray Crystallography; antimicrobial; antimicrobial drug; base; cell killing; comparative; design; endopeptidase Clp; flexibility; fungus; genetic regulatory protein; high throughput screening; inhibitor/antagonist; mRNA Differential Displays; methicillin resistant Staphylococcus aureus; microorganism; multi drug transporter; mutant; novel; protein degradation; resistant strain; response; small molecule libraries; therapeutic target; tool; unfoldase

This project has two main elements. The major effort involves 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 is 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.Peptidase activity of ClpP will be measured using a FRET peptide that yields an easily quantifiable fluorescence signal when cleaved. The assay requires readily available chemicals, a modified peptide that has been synthesized in our laboratory, and purified ClpP protease, which is prepared in our laboratory. A preliminary screen of a small chemical library has allowed optimization of the assay and has identified a few leads for further study. The large scale screening of over 300,000 compounds is underway. Initial hits in the screen will be validated at NCGC by a second round of screening involving timed assays in order to eliminate false positives resulting from intrinsic fluorescence of the test chemicals. Validated hits will then 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.Our laboratory has begun to construct strains of E. coli to test candidate compounds identified in the HTS. ADEPs do not penetrate the outer membrane of Gram-negative bacteria. In addition, ADEPs are substrates for the multidrug transporter, AcrAB, which rapidly eliminates many drugs from the cell. We have constructed a strain that has been deleted for acrAB and have introduced a plasmid that expresses an outer membrane protein that alters the permeability of the outer membrane and allows compounds to enter E. coli cells. These strains will be useful for comparing the relative cell permeability and effectiveness as antibiotics of candidate compounds and ADEPs against E. coli. We have also arranged with Dr. Scott Stibitz from Center for Biologics Evaluation, FDA, to test the compounds against several Gram positive and Gram-negative pathogenic bacterial strains and to evaluate the rate at which resistance arises. Resistance could result from mutations in ClpP that cause impaired binding of the active compounds or severely compromised ClpP activity. Both types of mutants should be rare because they would be expected to lack important biological activities of ClpXP and ClpA/C-P and thus compromise the growth of the bacteria in natural environments. Development of resistance by acquisition of mutations in the targets can be expected to be rare, because of the multiple targets of dysregulated ClpP, which must include precursors many vital enzymatic or regulatory proteins. To complement the efforts to identify new compounds that mimic ADEPs in their binding to ClpP and activating its protease activity, we have initiated a genetic screen to obtain mutants of ClpP that have altered binding properties and possibly altered allosteric responses to binding of ligands. 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 have developed a sensitive selection procedure that will identify mutants of ClpP that bind ADEPs less well but continue to bind ClpX and ClpA/C and thus retain biological function. The selection is based on the ability of ClpXP to degrade proteins that have an 11-amino acid tag (called an SsrA tag) at the C-terminus. By engineering the tag at the C-terminus of a toxic protein, we have created a strain that can only grow when ClpXP is functional within the cell. We have used a similar strategy based on a different toxic protein in the past to successfully isolate mutants of ClpX with altered substrate recognition properties (Erica N. Jones and Michael R. Maurizi, manuscript in preparation). We have modified the selection system in order to first allow screening of a plasmid library expressing mutated ClpP for resistance to ADEP. This initial screen must be done under conditions in which the toxic protein is tightly repressed. Once the library has been enriched for plasmids expressing ClpP that is not activated by ADEP (thus allowing cell survival in the presence of ADEP) we will induce the SsrA-tagged toxic protein and look for survivors that retain ClpP activity as evidenced by their ability to degrade the SsrA-tagged toxic protein. Forms of ClpP that appear to display differential binding to ADEPs and ClpX will be characterized further by standard biochemical assays. The mutated ClpP will also be tested for their sensitivity to candidate compounds identified in the HTS. 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.

该项目有两个主要要素。主要工作包括与 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)，以寻找与 ADEP 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。 ClpP 的肽酶活性将使用 FRET 肽进行测量，该肽在裂解时产生易于定量的荧光信号。该测定需要现成的化学品、我们实验室合成的修饰肽和我们实验室制备的纯化 ClpP 蛋白酶。对小型化学库的初步筛选可以优化检测方法，并确定了一些线索供进一步研究。超过 300,000 种化合物的大规模筛选正在进行中。 NCGC 将通过第二轮筛选（涉及定时测定）对筛选中的初始命中进行验证，以消除测试化学品固有荧光导致的假阳性。然后，经过验证的命中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 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我们的实验室已开始构建大肠杆菌菌株来测试 HTS 中确定的候选化合物。 ADEP 不能穿透革兰氏阴性细菌的外膜。此外，ADEP 是多药物转运蛋白 AcrAB 的底物，可快速消除细胞中的许多药物。我们构建了一种删除了 acrAB 的菌株，并引入了一种表达外膜蛋白的质粒，该蛋白可以改变外膜的通透性并允许化合物进入大肠杆菌细胞。这些菌株可用于比较候选化合物和 ADEP 对抗大肠杆菌的相对细胞渗透性和有效性。我们还与 FDA 生物制品评估中心的 Scott Stibitz 博士合作，测试这些化合物对几种革兰氏阳性和革兰氏阴性致病细菌菌株的作用，并评估耐药性产生的速度。 ClpP 突变可能导致耐药性，导致活性化合物的结合受损或 ClpP 活性严重受损。两种类型的突变体都应该很少见，因为它们缺乏 ClpXP 和 ClpA/C-P 的重要生物活性，从而损害细菌在自然环境中的生长。由于 ClpP 失调的多个靶点，其中必须包括许多重要的酶或调节蛋白的前体，因此通过靶点中获得突变而产生耐药性的情况预计是罕见的。为了补充鉴定模拟 ADEP 与 ClpP 结合并激活其蛋白酶活性的新化合物的努力，我们启动了遗传筛选以获得 ClpP 突变体，这些突变体改变了结合特性，并可能改变了对配体结合的变构反应。 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，可以识别与 ADEP 结合较差但继续结合 ClpX 和 ClpA/C 的 ClpP 突变体，从而保留生物学功能。该选择基于 ClpXP 降解 C 末端具有 11 个氨基酸标签（称为 SsrA 标签）的蛋白质的能力。通过在有毒蛋白的 C 末端设计标签，我们创建了一种只有当 ClpXP 在细胞内发挥功能时才能生长的菌株。我们过去曾使用基于不同毒性蛋白的类似策略，成功分离出具有改变的底物识别特性的 ClpX 突变体（Erica N. Jones 和 Michael R. Maurizi，手稿正在准备中）。我们修改了选择系统，以便首先筛选表达突变 ClpP 的 ADEP 抗性质粒库。该初始筛选必须在有毒蛋白质被严格抑制的条件下进行。一旦文库富集了表达不被 ADEP 激活的 ClpP 的质粒（从而允许细胞在 ADEP 存在的情况下存活），我们将诱导 SsrA 标记的毒性蛋白并寻找保留 ClpP 活性的幸存者，这通过它们的能力来证明降解 SsrA 标记的有毒蛋白。与 ADEP 和 ClpX 表现出差异结合的 ClpP 形式将通过标准生化测定进一步表征。突变的 ClpP 还将测试其对 HTS 中确定的候选化合物的敏感性。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。