Studies of the antibacterial activity of and resistance to molecules targeting the ClpP peptidase

ClpP 肽酶靶向分子的抗菌活性和耐药性研究

• 批准号: 10073398
• 负责人: Jason K Sello
• 金额: $ 51.17万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10073398
• 关键词: Studies; antibacterial; activity; resistance; molecules

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.

该提案概述了实验，这些实验将提供机制见解并加速 扰乱结核分枝杆菌 ClpP 肽酶功能的小分子的药物开发， 最致命的细菌病原体。 Mtb ClpP 肽酶是一种非典型复杂的桶状组合体 十四个亚基通过与 ATP 依赖性伴侣结合来降解蛋白质 识别并展开底物。在过去的五年里，我和我的合作者麻省理工学院的 Robert Sauer 教授 在异十四聚 ClpP 的功能重建上投入了大量的时间和精力 以及来自 Mtb 的辅助 ATP 酶（ClpX 和 ClpC1）。凭借这一成功，我们已经能够描述我们的 合理设计的 ClpP 活性调节剂。大量的初步数据（其中大部分已发布） 我们生成的结果为拟议的研究提供了清晰的路线图。他们将深入了解蛋白质 可用于药物开发的细菌的稳态以及可能急需的分子 用于对抗多重耐药结核病的日益减少的军备库的补充。 目标 1. 开发并表征 Mtb ClpP 系统的小分子调节剂。这个目标是 以化学合成和酶学为中心。一个目标是优化的多克合成， 抑制 ClpP 系统的杀分枝杆菌 ADEP，用于小鼠结核病模型。另一个是 优化新型 ADEP 片段，通过激活 Mtb 的 ClpP 系统来杀死 Mtb。我们还将综合 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（由我们的实验室开发）。化合物将在 Clp 系统活性的体外测定中进行评估。 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 目的是对分枝杆菌活培养物中 ClpP 调节剂的比较评估，包括 最小抑制和杀菌浓度的测定。我们相信，底物的鉴定 Mtb Clp 系统将揭示这些化合物的作用机制。通过应用新的蛋白质组学 技术以及成熟的耻垢分枝杆菌 Clp 体内 ClpP 底物捕获方法 系统首次，我们将在蛋白酶的天然状态下识别其底物，并且因为它受到以下因素的干扰 调制器。我们还将研究增强 ADEP 活性的小分子机制 Mtb 高达 16 倍（即假设的外流抑制剂和结核病药物贝达喹啉）。 目标 3. 评估 ClpP 调节剂在小鼠结核病模型中的活性。尽管 ADEP 已治愈 动物中的某些细菌感染，尚未在结核病中进行研究。在小鼠模型中 针对结核病，约翰·霍普金斯大学的 William Bishai 博士将单独评估优化的 ADEP 的功效以及在 与贝达喹啉、标准结核病药物治疗方案以及我们设计的增强剂组合 通过 Mtb 抑制 ADEP 外流。我们将使用我们的 Mtb ClpP 激活剂进行类似的实验。