Determination of the Biological Roles and Chemical Mechanisms of the Glutamate Ra

描述（由申请人提供）：本R21申请中提出的研究解决了开发新型抗生素的迫切需求，这些抗生素针对新出现的感染性病原体，以及有可能用作生物武器的致病菌。本研究的长期目标是开发一类新的谷氨酸外消旋酶抑制剂，该抑制剂可催化L-谷氨酸向d -谷氨酸的立体转化，而d -谷氨酸是细胞壁生物合成的重要代谢物。谷氨酸外消旋酶在几种细菌中是必需的，但在哺乳动物中没有发现，因此被预测为极好的抗生素靶点。值得注意的是，目前至少有三家制药公司正在开发谷氨酸外消旋酶抑制剂作为潜在的抗菌药物，从而支持谷氨酸外消旋酶作为抗菌靶点的潜在重要性。然而，我们的抑制剂设计策略与这些公司完全不同，并且基于谷氨酸外消旋酶的过渡态结构，我们预测与基于基态酶-底物复合物的药物相比，谷氨酸外消旋酶与酶的结合具有更高的亲和力。在这个R21申请中，我们提出探索性研究的目标是：(i)在体外和体内表征两种炭疽芽孢杆菌谷氨酸外消旋酶RacE1和RacE2的重要性和性质，以及（ii）建立两种酶催化反应的过渡态结构模型。这个高度跨学科的应用程序整合了生物有机和计算化学，生物化学和细菌发病机理方面的大量专业知识。具体目标是：描述racE1和racE2的重要性和作用。具体目标2。表征RacE1-和race2催化的外消旋化的过渡态。这些特定目标的预期结果将是验证谷氨酸外消旋酶作为炭疽芽胞杆菌的药物靶点，并建立RacE1和RacE2的过渡状态模型。从这些模型中，我们将确定小分子过渡态类似物，将筛选对RacE1和/或RacE2酶活性的抑制活性。这些研究的结果将为未来的工作提供实验和概念框架，以优化小分子“先导物”，使其成为具有抗菌活性的超特异性反应型抑制剂。公共卫生相关性：该申请解决了现有的和迫切的需要，即开发针对新出现的传染性病原体的新型抗生素，以及那些可能被用作生物武器的病原体。这些研究的完成将导致对炭疽杆菌（炭疽杆菌）具有潜在抗菌活性的新型抑制剂。由该基金支持的工作开发的方法也可能适用于开发针对其他生物医学上重要致病菌的抗生素。

DESCRIPTION (provided by applicant): Studies proposed in this R21 application address the urgent need to develop new classes of antibiotics against emerging infectious agents, as well as pathogenic bacteria with the potential to be used as bio-weapons. The long-term goal of the work proposed in this application is to develop a new class of inhibitors against glutamate racemases, which catalyze the stereo-inversion of L- to D-glutamate, an important metabolite for cell wall biosynthesis. Glutamate racemases are essential in several bacteria, but not found in mammals, and are thus predicted to be excellent antibiotic targets. Notably, at least three pharmaceutical companies are currently developing glutamate racemase inhibitors as potential antimicrobial drugs, thereby supporting the potential importance of the glutamate racemases as antimicrobial targets. However, our strategy for inhibitor design is entirely different than these companies, and is based on the transition state structure of glutamate racemase, which we predict will bind to the enzyme with higher affinity than do drugs based on the ground state enzyme-substrate complexes. In this R21 application, we propose exploratory studies towards the goals of (i) characterizing the importance and properties of the two Bacillus anthracis glutamate racemases, RacE1 and RacE2, in vitro and in vivo, and, (ii) generating models of the transition state structures of the reactions catalyzed by both enzymes. This highly interdisciplinary application consolidates considerable expertise in bioorganic and computational chemistry, biochemistry, and bacterial pathogenesis. The Specific Aims are: Specific Aim 1. To characterize the importance and roles of racE1 and racE2. Specific Aim 2. To characterize the transition states of RacE1- and RacE2-catalyzed racemization. The anticipated outcomes of these specific aims will be validation of glutamate racemase as a drug-target in B. anthracis, and the generation of transition state models for both RacE1 and RacE2. From these models, we will identify small molecule transition state analogs that will be screened for inhibitory activities against RacE1 and/or RacE2 enzyme activities. The results from these studies will provide the experimental and conceptual framework for future work to optimize small molecule "leads" into ultra-specific, reaction-based inhibitors with antimicrobial activity. PUBLIC HEALTH RELEVANCE: This application addressed an existing and urgent need to develop new classes of antibiotics against emerging infectious agents, as well as those agents that may potentially be used as bio-weapons. Completion of these studies will result in a new class of inhibitors with potential antimicrobial activity against Bacillus anthracis, which causes anthrax. The methodologies developed by work supported by this grant will also be potentially applicable to the development of antibiotics against other biomedically important pathogenic bacteria.