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

这个项目有两个主要要素。这项主要工作始于两年前，涉及与美国国立卫生研究院化学基因组学中心(NCGC)的科学家合作，对一个大型化学库进行高通量筛选(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。这个项目的部分资金来自2012年授予我的R03奖(1 R03 MH095569)。X射线结晶学表明，ADEP和ClpP之间的相互作用表明，很有可能找到显示模拟ADEP的芳香/脂肪部分的刚性结构的有机分子，对接到ClpP，并对其活性施加变构效应。ADEP和ClpP之间的主要接触涉及ADEP中的一个芳环与ClpP顶端表面的一个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轴向通道延伸的疏水沟槽之间存在疏水相互作用。其他次要的相互作用包括与ADEP短肽段的主干原子的氢键结合。ADEP的脱脂多肽部分与ClpP相互作用很少，主要用于限制ADEP中脂肪区域的构象灵活性，这些脂肪区域固定在锁定到对接位置的配置中。ADEP的溶液结构本身证实，当与ClpP结合时，其诱导的变化很小。经过对30多万个化合物的大规模筛选，约18个化合物被鉴定为潜在的ClpP抑制剂，约30个化合物被鉴定为潜在的激活剂。这些化合物现在正在接受更详细的测试，以确定它们对ClpP的各种活性的影响。被确认为有效的抑制剂激活剂的化合物将被大量提供，以供进一步研究和结构研究，以确定结合的位置和方式。它们将在我的实验室中进行进一步的检测，以获得更完整的结合亲和力、对多肽和蛋白质底物的激活作用以及对人、大肠杆菌和枯草杆菌CLPP的相对特异性。然后，将测试化合物对实验室菌株大肠杆菌和枯草杆菌的抗菌活性。化合物还将测试其对几种人类癌细胞株的生长抑制活性。一旦通过上述各种二次分析确定并筛选出有希望的先导化合物，NCGC的合成化学团队将开始设计合成策略，以制造这些化合物和化合物的变体，以开发新的版本，这些版本针对与ClpP的结合和对抗细菌培养的有效性进行了优化。为了补充寻找与ClpP结合时模仿ADEP的新化合物的努力，我们进行了遗传筛选，以获得改变了结合性质的ClpP突变体，并可能改变了对ADEP结合的变构反应。ADEP结合到ClpX和ClpA/C所使用的ClpP顶端表面的对接位置，形成具有生物功能的ClpXP和ClpAP复合体。我们开发了一种灵敏的选择程序来鉴定对ADEP具有抗性但与ClpX保持酶活性的ClpP突变体。这一选择是基于ClpXP在C末端降解带有11个氨基酸的降解标签(称为SsrA标签)的蛋白质的能力。从一组多重突变的ClpP中，我们分离到了六种形式的带有单一突变的ClpP。表达突变体的细胞保持了降解SsrA标记蛋白的活性，并对ADEP有不同程度的抗性。我们已经纯化了突变蛋白，目前正在研究它们的体外生化和酶活性。本工作的目的是确定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的变构反应，导致通道扩张并允许蛋白质不分青红皂白地进入。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，从而产生新的相互作用，从而稳定ClpP的激活结构。在一项相关的工作中，我们已经开始了合成ClpP的β-内酯抑制剂的努力。最初，我们正在制造两种文献中描述的抑制剂，并计划对过程进行修改，以引入其他取代基，这些取代基应该有助于增加与ClpP的结合亲和力。这些抑制剂将与纯化的ClpP反应，研究它们对四级结构的影响，并获得晶体结构数据，以阐明它们是如何结合到ClpP活性部位的。

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.