Discovery of inhibitors that target the Mtb ClpP1P2 protease

项目摘要 作为“蛋白质生产和降解的调节作为一种综合方法”方案的一部分 对药物敏感和耐药的结核分枝杆菌(结核分枝杆菌)的快速灭菌“，项目2将侧重于 “发现针对Mtb ClpP1P2蛋白水解酶的抑制剂”。遗传学研究表明，CLP系统 代表了一种独特的有吸引力的药物靶点。它在所有条件下都是必不可少的，无论是增长的还是非增长的。 而且它受到严格的调控--即使是活动的微小变化也会导致细胞死亡。当CLP蛋白耗尽时， 无论是在体外还是在结核病的小鼠模型中，细胞都会迅速死亡。事实上，死亡的速度比现在快得多 伴随着传统药物靶点的枯竭。因此，针对CLP的抗生素可以提供一条通往 抢手的治疗--缩短疗程。 已经发现了几种针对CLP复合体的化合物。其中绝大多数化合物 以ATPase组分之一ClpC1为目标，这些化合物将作为项目1的一部分进行优化。 相反，项目2将专注于ClpP1P2蛋白酶的调节器，这是CLP的核心功能。在其他细菌中， CLP蛋白分解活性的激活剂是致命的。遗传学研究表明，抑制CLP蛋白酶在 分枝杆菌也会导致细胞死亡。此外，一系列修饰的多肽抑制CLP介导的蛋白降解 并以CLP依赖的方式体外杀伤结核分枝杆菌。因此，项目2的目标是与所有 开发这些有希望的化合物以提高其活性和药理作用的科学核心，朝着 该计划的最终目标是开发新的抗结核药物。特别是，该项目将与核心一起工作 A和C帮助设计和合成新的抑制剂，并依赖核心B和D进行测试和生产 发展。具体而言，项目2将： 1.鉴定CLP抑制肽的序列决定因素。序列的映射 CLP蛋白水解酶的专一性已被用于设计第一代抑制剂。该项目将使用提纯的 ClpP1P2复合体在体外进一步确定多肽特性，使设计改进的抑制剂成为可能。 2.开发优化的基于机理的多肽抑制剂。该项目将使用来自 底物鉴定，以产生作为抑制剂的修饰多肽。这些将首先进行测试，以确定其 抑制酶的能力，然后是杀死结核分枝杆菌的能力。对于那些最理想的，从药理和 毒理学参数将被确定为药物开发的第一步。 3.确定作为CLP抑制剂的非肽类似物。多肽通常具有药理作用 可能使它们变得非最佳的问题。非肽类抑制剂将使用优化的底物进行设计 和共晶体。这些都将用纯化的酶和全细胞分析进行测试。

Project Summary As part of the program “Modulation of Protein Production and Degradation as an Integrated Approach to Rapid Sterilization of Drug Sensitive and Resistant Mycobacterium tuberculosis (Mtb)”, Project 2 will focus on “Discovery of inhibitors that target the Mtb ClpP1P2 protease”. Genetic studies suggest that the Clp system represents a uniquely attractive drug target. It is essential under all conditions, both growing and non-growing. And it is tightly regulated – even small changes in activity result in cell death. Upon depletion of Clp proteins, cells rapidly die, both in vitro and in a mouse model of tuberculosis. In fact, the death is much more rapid than is seen with depletion of traditional drug targets. Thus, an antibiotic that targeted Clp could provide a path to a sought-after treatment-shortening regimen. Several compounds have been found that target the Clp complex. The vast majority of those compounds target one of the ATPase components, ClpC1, and these compounds will be optimized as part of Project 1. Project 2 will instead focus on modulators of the ClpP1P2 protease, the core function of Clp. In other bacteria, activators of Clp proteolytic activity are lethal. Genetic studies have shown that inhibition of Clp protease in mycobacteria also results in cell death. Moreover, a series of modified peptides inhibit Clp-mediated proteolysis in vitro and kill Mtb in a Clp-dependent manner. Thus, the goal of Project 2 is to work together with all the Scientific Cores to develop these promising compounds to improve their activity and pharmacology, towards the program’s ultimate goal of developing new antituberculous agents. In particular, the project will work with Cores A and C to help design and synthesize new inhibitors, and rely on Cores B and D for testing and product development. Specifically, Project 2 will: 1. Identify the sequence determinants of Clp-inhibitory peptides. Mapping of the sequence specificity of the Clp protease has been used to design first generation inhibitors. This project will use purified ClpP1P2 complexes in vitro to further define peptide characteristics to enable design of improved inhibitors. 2. Develop optimized mechanism-based peptide inhibitors. The project will use information from substrate identification to produce modified peptides that act as inhibitors. These will initially be tested for their ability to inhibit the enzyme and then their ability to kill Mtb. For those that are optimal, pharmacologic and toxicologic parameters will be determined as an initial step toward drug development. 3. Identify non-peptidic analogues that act as Clp inhibitors. Peptides often have pharmacologic issues that could make them non-optimal. Non-peptidic inhibitors will be designed using optimized substrates and co-crystals. These will be tested with both purified enzymes and in whole cell assays.