Understanding the impact of Antigen 85 complex substrate specificity on mycobacte

了解抗原 85 复合物底物特异性对分枝杆菌的影响

• 批准号: 7940613
• 负责人: Donald R Ronning
• 金额: $ 40.92万
• 依托单位: UNIVERSITY OF TOLEDO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7940613
• 关键词: AG 85; Active Sites; Address; Affect; Affinity; Antigens; Bacteria; Binding; Biochemical; Biochemical Reaction; Biological Assay; Calorimetry; Catalysis; Complex; Cord Factors; Data; Development; Disease; Enzymes; Future; Genus Mycobacterium; Goals; Homologous Gene; Immune system; In Vitro; Infection; Kinetics; Lead; Measurement; Measures; Methods; Molecular Conformation; Movement; Mutation; Mycobacterium tuberculosis; Opportunistic Infections; Pharmaceutical Preparations; Play; Preclinical Drug Evaluation; Reaction; Research Project Grants; Role; Serine; Shapes; Specificity; Structural Protein; Structure; Substrate Interaction; Substrate Specificity; Techniques; Testing; Thermodynamics; Titrations; Trehalose; Tuberculosis; Variant; X-Ray Crystallography; acyl group; arabinogalactan; base; cell envelope; design; drug development; in vivo; inhibitor/antagonist; interest; killings; mutant; mycobacterial; novel; novel therapeutics; pathogenic bacteria; preference; public health relevance; research study; tuberculosis treatment

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.

描述（由申请方提供）：了解结核分枝杆菌（mtb）抗原85酶与其底物之间的相互作用，鉴别导致底物特异性差异的每种酶组分，鉴别催化活性抑制剂，并结合这些数据联合收割机开发用于药物开发的新型先导化合物。抗原85（Ag 85）酶催化合成分枝杆菌的高度疏水性细胞包膜所需的最后步骤。体内研究表明MTB的三种Ag 85酶负责合成不同的细胞包膜组分，海藻糖二霉菌酸酯（TDM）或霉菌酰-阿拉伯半乳聚糖（mAG）。然而，尚不清楚三种Ag 85同系物如何实现这种底物特异性和选择性。在这个建议中，我们结合联合收割机生物化学，生物物理和结构的方法来解决这个问题。这个研究项目分为四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定量抗原85酶与其底物之间的酶促反应和结合相互作用。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射线晶体学直接可视化催化过程中酶中发生的结构变化。通过获得反应中每个步骤的这些快照，我们可以获得关于抗原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中突变的影响，以探测结构变化对酶促反应的影响以及与这些酶所使用的不同底物的相互作用。稳态动力学，结合研究，和X射线晶体学都将被用来检查这些突变体。最后一种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复合物形成的热力学，并允许比较三种抗原85酶。其次，这些化合物代表了开发治疗结核病、麻风病、M.溃疡分枝杆菌的机会性感染;免疫系统受到抑制的人。 公共卫生相关性：结核病在全世界造成的死亡人数超过任何其他可治疗的疾病。典型的药物治疗持续6个月。迫切需要更好的结核病治疗药物。了解这些致病菌中的酶功能对于开发特异性抑制这些酶并杀死细菌的新药至关重要。这项研究试图更好地了解3种结核病酶的功能，这些酶对于创造导致结核病的细菌的保护性外层非常重要，这是设计新的和更好的治疗结核病药物的基础。

项目成果

Targeting the mycobacterial envelope for tuberculosis drug development.

• DOI: 10.1586/eri.12.91
• 发表时间: 2012-09
• 期刊: Expert review of anti-infective therapy
• 影响因子: 5.7
• 作者: Favrot L;Ronning DR
• 通讯作者: Ronning DR

Zafirlukast inhibits complexation of Lsr2 with DNA and growth of Mycobacterium tuberculosis.

Zafirlukast 抑制 Lsr2 与 DNA 的复合以及结核分枝杆菌的生长。

• DOI: 10.1128/aac.02407-12
• 发表时间: 2013
• 期刊: Antimicrobial agents and chemotherapy
• 影响因子: 4.9
• 作者: Pinault,Lucile;Han,Jeong-Sun;Kang,Choong-Min;Franco,Jimmy;Ronning,DonaldR
• 通讯作者: Ronning,DonaldR

Design, synthesis, and X-ray analysis of a glycoconjugate bound to Mycobacterium tuberculosis antigen 85C.

与结核分枝杆菌抗原 85C 结合的糖复合物的设计、合成和 X 射线分析。

• DOI: 10.1021/bc3004342
• 发表时间: 2012-12-19
• 期刊: BIOCONJUGATE CHEMISTRY
• 影响因子: 4.7
• 作者: Ibrahim, Diaa A.;Boucau, Julie;Lajiness, Daniel H.;Veleti, Sri Kumar;Trabbic, Kevin R.;Adams, Samuel S.;Ronning, Donald R.;Sucheck, Steven J.
• 通讯作者: Sucheck, Steven J.