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.

这项提案概述了实验，这些实验将提供机械性的见解并加速 干扰结核分枝杆菌ClpP多肽酶功能的小分子药物开发， 最致命的细菌病原体。Mtb ClpP多肽酶是一个非典型的复杂的桶形组合 在14个亚基中，通过与依赖于ATP的伴侣蛋白结合来降解蛋白质 识别并展开衬底。在过去的五年里，我和我的合作者，麻省理工学院的罗伯特·鲍尔教授 在异十四聚体ClpP的功能重建上投入了大量的时间和精力 和来自Mtb的附件ATPase(ClpX和ClpC1)。凭借这一成功，我们能够将我们的 合理设计ClpP活性调节剂。大量的初步数据(其中大部分已经公布) 我们所产生的结果为拟议的研究提供了一个明确的路线图。他们将对蛋白质有深入的了解 可用于药物开发的细菌的动态平衡和可能非常需要的分子 增加了用于对抗多重耐药结核杆菌的日益减少的医疗设备。 目的1.开发和表征Mtb ClpP体系的小分子调节剂。这个目标是 以化学合成和酶学为中心。一个目标是多克合成优化的， 用于小鼠结核病模型的抑制ClpP系统的分枝杆菌杀菌剂ADEP。另一个是 一种通过激活其ClpP系统杀死结核分枝杆菌的新型ADEP片段的优化。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作用 抑制剂(在我们的实验室开发)。化合物将在体外CLP系统活性测试中进行评估。 目的2.评估分枝杆菌活培养物中ClpP调节剂的活性和抗药性。这 AIM专注于对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，对其底物的鉴定 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 污垢分枝杆菌体内成熟的ClpP底物捕捉法 系统第一次，我们将确定它的底物在蛋白水解酶的天然状态和当它被扰动 调制器。我们还将研究小分子增强ADEP活性的机制。 结核分枝杆菌的抑制率高达16倍(即假想的外排抑制剂和结核病药物贝达奎林)。 目的3.评估ClpP调节剂在小鼠结核病模型中的活性。虽然ADEP治愈了 在动物中的某些细菌感染，它们还没有在结核病中进行研究。在小鼠模型中 约翰·霍普金斯大学的威廉·比沙伊博士将评估优化的ADEP单独和在 与贝达奎林、标准结核病药物方案和我们设计的增效剂联合使用 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。