Understanding the impact of Antigen 85 complex substrate specificity on mycobacte

描述（由申请人提供）：了解结核分枝杆菌（mtb）抗原85酶与其底物之间的相互作用，鉴定导致底物特异性差异的每种酶的成分，鉴定催化活性抑制剂，并结合这些数据开发用于药物开发的新型先导化合物。抗原85 （Ag85）酶催化了分枝杆菌高度疏水细胞包膜合成所需的最后一步。体内研究表明，mtb的三种Ag85酶负责合成不同的细胞包膜成分，海藻糖二真菌酸（TDM）或真菌酸-阿拉伯半乳糖（mAG）。然而，目前尚不清楚三种Ag85同源物是如何实现这种底物特异性和选择性的。在这个建议中，我们结合了生化、生物物理和结构方法来解决这个问题。本研究项目分为4个独立但相互关联的方面。首先，我们将使用稳态动力学和等温滴定量热法来量化酶促反应和抗原85酶与其底物之间的结合相互作用。这将进一步完善我们对这三种酶在细菌中发挥的不同作用的理解。第二组实验使用x射线晶体学直接观察酶在催化过程中发生的结构变化。通过获得反应中每个步骤的快照，我们可以获得抗原85/底物相互作用的前所未有的细节。本研究的第三部分考察了酶突变的影响，以探索结构变化对酶反应的影响以及与这些酶使用的不同底物的相互作用。稳态动力学、结合研究和x射线晶体学都将用于检查这些突变体。最后的研究方法是开发抗原85酶的新型抑制剂。这将带来两个好处。首先，这些化合物将用于研究络合物形成的热力学，并允许比较三种抗原85酶。其次，这些化合物为开发治疗结核病、麻风病、溃疡分枝杆菌感染和免疫系统受到抑制的人的鸟分枝杆菌机会性感染的新疗法提供了线索。

DESCRIPTION (provided by applicant): To understand the interactions between the Mycobacterium tuberculosis (mtb) antigen 85 enzymes and their substrates, identify components of each enzyme that lead to differences in substrate specificity, identify inhibitors of the catalytic activity, and combine these data to develop novel lead compounds for drug development. The antigen 85 (Ag85) enzymes catalyze the final step required for synthesis of the highly hydrophobic cell envelope of mycobacteria. In vivo studies suggest the three Ag85 enzymes of mtb are responsible for synthesizing different cell envelope components, trehalose dimycolate (TDM) or mycolyl-arabinogalactan (mAG). However, it is not clear how the three Ag85 homologs achieve this substrate specificity and selectivity. In this proposal, we combine biochemical, biophysical, and structural approaches to address this question. This research project is separated into 4 separate but interrelated aspects. First, we will use steady-state kinetics and isothermal titration calorimetry to quantify the enzymatic reaction and binding interactions between the antigen 85 enzymes and their substrates. This will further refine our understanding the different roles these three enzymes play in the bacterium. The second set of experiments uses X-ray crystallography to directly visualize structural changes that occur in the enzymes during catalysis. By obtaining these snapshots of each step in the reaction, we can obtain unprecedented detail about the antigen 85/substrate interactions. The third portion of this study examines the affect of mutations in the enzymes to probe the affects of structural changes on the enzymatic reaction and interactions with the different substrates used by these enzymes. Steady-state kinetics, binding studies, and X-ray crystallography will all be used to examine these mutants. The final method of inquiry will develop novel inhibitors of the antigen 85 enzymes. This will offer two benefits. First, these compounds will be used to study the thermodynamics of complex formation and allow comparison of the three antigen 85 enzymes. Second, these compound represent leads for the development of new therapeutics for the treatment of tuberculosis, lepresy, M. ulcerans infections, and the opportunistic infection by M. avium specied of people with suppressed immune systems. PUBLIC HEALTH RELEVANCE: Tuberculosis kills more people worldwide than any other treatable disease. Typical treatment with drugs lasts 6 months. Better drugs for treating tuberculosis are desperately needed. Understanding enzyme function in these pathogenic bacteria is central to developing new drugs that specifically inhibit those enzymes and kill the bacteria. This study attempts to better understand the function of 3 tuberculosis enzymes that are important for creating the protective outer layer of the bacteria that cause tuberculosis as a basis for designing new and better drugs to treat tuberculosis.