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: 8763529
• 负责人: MICHAEL MAURIZI
• 金额: $ 25.24万
• 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/8763529
• 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 Targeting; 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; 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; screening; 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.

这个项目有两个主要元素。主要工作包括与NIH化学基因组学中心（NCGC）的科学家合作，对一个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。本项目部分资金来源于2012年授予我的R03奖（1 R03 MH095569）。x射线晶体学显示，ADEP和ClpP之间的相互作用表明，应该很有可能找到具有刚性结构的有机分子，模仿ADEP的芳香族/脂肪族部分，与ClpP对接，并对其活性施加变构作用。ADEP和ClpP之间的主要接触涉及ADEP中的芳香环与ClpP顶端表面的深袋之间的疏水相互作用。此外，在ADEP中的脂肪链和从疏水袋向ClpP的轴向通道延伸的疏水沟之间存在疏水相互作用。其他次要的相互作用包括氢结合，涉及来自ADEP短肽段的主链原子。ADEP的沉积肽部分与ClpP的相互作用很小，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单独的溶液结构证实其与ClpP结合后几乎没有诱导变化。ClpP的肽酶活性将使用FRET肽来测量，该肽在裂解时产生易于量化的荧光信号。该试验需要现成的化学物质，在我们实验室合成的修饰肽，以及在我们实验室制备的纯化的ClpP蛋白酶。对一个小的化学文库进行初步筛选，优化了该分析方法，并确定了一些可供进一步研究的线索。超过30万种化合物的大规模筛选正在进行中。初始筛选结果将在NCGC通过第二轮筛选进行验证，包括定时分析，以消除由测试化学品的固有荧光引起的假阳性。然后将在我的实验室进一步分析验证的片段，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。一旦有希望的先导化合物通过上述各种二级分析被识别和筛选，NCGC的合成化学团队将开始设计合成策略，以制造化合物和化合物的变体，以开发新的版本，以优化与ClpP的结合，并有效对抗细菌培养。我们的实验室已经开始构建大肠杆菌菌株，以测试在HTS中发现的候选化合物。adep不能穿透革兰氏阴性菌的外膜。此外，adep是多药物转运体AcrAB的底物，AcrAB可以迅速清除细胞中的许多药物。我们构建了一株被acrAB删除的菌株，并引入了一种表达外膜蛋白的质粒，该质粒可以改变外膜的通透性，并允许化合物进入大肠杆菌细胞。这些菌株将有助于比较候选化合物和adep对大肠杆菌的相对细胞通透性和作为抗生素的有效性。我们还与FDA生物制品评估中心的Scott Stibitz博士进行了安排，对几种革兰氏阳性和革兰氏阴性致病菌菌株进行了测试，并评估了耐药性产生的速度。耐药可能是由于ClpP突变导致活性化合物结合受损或ClpP活性严重受损。这两种类型的突变体应该是罕见的，因为它们可能缺乏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。由于失调的ClpP有多个靶标，其中必须包括许多重要酶或调节蛋白的前体，因此通过在靶标中获得突变而产生耐药性的可能性很小。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，我们启动了一项基因筛选，以获得改变结合特性和可能改变配体结合变构反应的ClpP突变体。adep结合到ClpX和ClpA/C使用的ClpP顶端表面的对接位点，形成具有生物学功能的ClpXP和ClpAP复合物。我们开发了一种敏感的选择程序，可以识别结合adep较差但继续结合ClpX和ClpA/C并因此保留生物功能的ClpP突变体。这种选择是基于ClpXP降解在c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端设计标签，我们创造了一种只有当ClpXP在细胞内起作用时才能生长的菌株。过去，我们使用了基于不同毒性蛋白的类似策略，成功分离出具有改变底物识别特性的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的质粒库以对ADEP产生抗性。初始筛选必须在有毒蛋白被严格抑制的条件下进行。一旦文库丰富了表达未被ADEP激活的ClpP的质粒（因此允许细胞在ADEP存在下存活），我们将诱导ssra标记的有毒蛋白，并寻找保留ClpP活性的幸存者，这可以通过它们降解ssra标记的有毒蛋白的能力来证明。与adep和ClpX表现出差异结合的ClpP形式将通过标准生化分析进一步表征。突变的ClpP还将测试它们对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中涉及adep和ClpX结合的关键残基，以及与轴向通道通信并导致通道扩展并允许不加区分的蛋白质进入的变弹性反应。对ADEP和ClpX反应不同的突变形式的ClpP可能表现出不同的结合亲和力或结合率，或者可能受到残基的影响，这些残基产生新的相互作用，从而稳定了活化的ClpP结构。