Studies of the Antibacterial Activity of and Resistance to Molecules Targeting the ClpP Peptidase

ClpP肽酶分子的抗菌活性和耐药性研究

• 批准号: 9755347
• 负责人: Jason K Sello
• 金额: $ 11.96万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-25 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9755347
• 关键词: ATP phosphohydrolase; Animals; Anti-Bacterial Agents; Bacteria; Bacterial Infections; Bacterial Proteins; Binding; Biological Assay; Complex; Core Facility; Curiosities; Data; Development; Drug Targeting; Drug resistance in tuberculosis; Enzymatic Biochemistry; Enzymes; Evaluation; Genetic; Genus Mycobacterium; Human; In Vitro; Investments; Laboratories; Lactones; Listeria monocytogenes; Methodology; Methods; Molecular Chaperones; Multi-Drug Resistance; Mycobacterium smegmatis; Mycobacterium tuberculosis; Pathogenicity; Peptide Hydrolases; Periodicity; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Physiological; Physiology; Production; Proteins; Proteome; Proteomics; Public Health Schools; Publications; Publishing; Regimen; Reporting; Research; Resistance; Role; Staphylococcus aureus; Streptococcus pneumoniae; Structure; System; Technology; Time; Tuberculosis; Universities; Virulence; Virulence Factors; bactericide; base; biological adaptation to stress; chemical synthesis; comparative; design; drug development; enzyme activity; experimental study; fight against; genetic approach; in vitro Assay; in vivo; inhibitor/antagonist; insight; mouse model; novel; optimism; pathogen; pathogenic bacteria; proteostasis; reconstitution; small molecule; success; treatment strategy; tuberculosis drugs; tuberculosis treatment

This proposal outlines experiments that will provide mechanistic insights into and accelerate the medicinal development of small molecules that perturb the function of the ClpP peptidase in M. tuberculosis, the deadliest bacterial pathogen. The Mtb ClpP peptidase is an atypically complex, barrel-shaped assemblage of fourteen subunits that degrades proteins through associations with ATP-dependent chaperones that recognize and unfold substrates. In the past five years, my collaborator, Prof. Robert Sauer at MIT and I have made significant investments of time and effort in the functional reconstitution of the heterotetradecameric ClpP and accessory ATPases (ClpX and ClpC1) from Mtb. With that success, we have been able to characterize our rationally designed modulators of ClpP activity. The voluminous preliminary data (much of which is published) that we have generated provide a clear roadmap for the proposed research. They will yield insights into protein homeostasis in bacteria that can be exploited in drug development and molecules that could be much needed additions to the dwindling armamentarium used in the fight against multi-drug resistant Mtb. Aim 1. Develop and characterize small molecule modulators of the Mtb ClpP system. This aim is centered in chemical synthesis and enzymology. One objective is the multi-gram synthesis of an optimized, mycobactericidal ADEP that inhibits the ClpP system for use in murine models of tuberculosis. Another is optimization of a novel ADEP fragment that kills Mtb by activation of its ClpP system. We will also synthesize rationally designed ClpP inhibitors lacking the pharmacological liabilities of the only known mechanism-based inhibitor (developed in our laboratories). Compounds will be evaluated in in vitro assays of Clp system activity. Aim 2. Assess activities of and resistance to ClpP modulators in living cultures of mycobacteria. This aim is focused on comparative evaluations of ClpP modulators in living cultures of mycobacteria- including assays of minimal inhibitory and bactericidal concentrations. We believe that identification of the substrates of the Mtb Clp system will reveal insights into the mechanisms of these compounds. By applying new proteomics technologies along with a well-established in vivo ClpP substrate trapping method to the M. smegmatis Clp system for the first time, we will identify its substrates in the protease's native state and as it is perturbed by modulators. We will also investigate the mechanisms of small molecules that potentiate ADEP activity against Mtb by as much as 16-fold (i.e., hypothetical suppressors of efflux and the tuberculosis drug bedaquiline). Aim 3. Assess activities of ClpP modulators in mouse models of tuberculosis. Though ADEPs cured certain bacterial infections in animals, they have not been studied in tuberculosis. In murine models of tuberculosis, Dr. William Bishai at Johns Hopkins will assess the efficacies of an optimized ADEP alone and in combination with bedaquiline, standard tuberculosis drug regimens, and a potentiator that we designed to act by suppression of ADEP efflux by Mtb. Analogous experiments will be performed with our Mtb ClpP activator.

该提案概述了将提供机械见解并加速 干扰M中ClpP肽酶功能的小分子的药物开发。肺结核， 最致命的细菌病原体Mtb ClpP肽酶是一种结构复杂的桶形集合体 由14个亚基组成，通过与ATP依赖性分子伴侣的结合降解蛋白质， 识别并展开基板。在过去的五年里，我的合作者，麻省理工学院的罗伯特·绍尔教授和我 在异十四聚体ClpP的功能重建方面投入了大量的时间和精力 和来自Mtb的辅助ATP酶（ClpX和ClpC 1）。有了这一成功，我们能够描述我们的 合理设计的ClpP活性调节剂。大量的初步数据（其中大部分已公布） 为拟议的研究提供了清晰的路线图。它们将使我们深入了解蛋白质 细菌中的稳态可以用于药物开发和可能非常需要的分子 增加了用于对抗多药耐药结核病的日益减少的医疗设备。 目标1.开发和表征Mtb ClpP系统的小分子调节剂。该目的是 以化学合成和酶学为中心。一个目标是优化的， 本发明涉及用于结核病鼠模型的抑制ClpP系统的杀分枝杆菌ADEP。另一个是 优化通过激活其ClpP系统杀死Mtb的新型ADEP片段。我们还将合成 合理设计的ClpP抑制剂缺乏唯一已知的基于机制的药理学责任， 抑制剂（在我们的实验室开发）。将在Clp系统活性的体外测定中评价化合物。 目标2.评估分枝杆菌活培养物中ClpP调节剂的活性和耐药性。这 目的是集中在分枝杆菌活培养物中ClpP调节剂的比较评估-包括 最小抑菌浓度和杀菌浓度的测定。我们认为，确定底物的 Mtb Clp系统将揭示这些化合物的机理。通过应用新的蛋白质组学 技术沿着与完善的体内ClpP底物捕获方法一起用于M.包皮垢 系统的第一次，我们将确定其底物在蛋白酶的天然状态，因为它是扰动 调制器。我们还将研究增强ADEP活性的小分子的机制， 结核分枝杆菌高达16倍（即，假设的外排抑制剂和结核病药物贝达喹啉）。 目标3.评估ClpP调节剂在结核病小鼠模型中的活性。虽然ADEP治愈 某些细菌感染的动物，他们还没有研究结核病。鼠类模型中 约翰霍普金斯的William Bishai博士将评估优化的ADEP单独和联合 与贝达喹啉、标准结核病药物方案和我们设计的增效剂联合作用， 通过Mtb抑制ADEP流出。类似的实验将用我们的Mtb ClpP激活剂进行。