Characterization of two acyl-CoA carboxylase complexes of Mycobacterium tuberculo

描述(申请人提供)：结核分枝杆菌是人类结核病(TB)的病原体，每年导致全球约300万人死亡，估计感染了世界三分之一的人口。由于缺乏药物依从性、多药耐药(MDR)菌株的出现以及艾滋病的流行而导致的这种疾病的再次出现，促使开发具有抗MDR菌株活性的新药，以缩短目前治疗的持续时间和复杂性。酰辅酶A羧化酶(ACCase)在大多数生物体内的脂肪酸代谢中起着至关重要的作用，已被认为是开发新型抗菌剂的良好靶点。在结核分枝杆菌中，从基因组序列中预测了6个可能的ACCase，其中两个ACCase4和ACCase6被认为参与了霉菌酸的生物合成。这种复杂的脂质是结核分枝杆菌细胞膜的主要成分之一，与这种细菌的生存和致病性有关。这项研究的目的是从遗传、生化和结构水平上描述结核分枝杆菌ACCases4和6的特征，并通过使用耻垢分枝杆菌作为工作模型，了解它们在霉菌酸生物合成和这些微生物生存中的生理作用和相关性。此外，我们将通过化学文库的电子对接在体内和体外表征可能的ACCase抑制剂，并利用它们来验证这些酶复合体作为未来抗分枝杆菌药物开发的靶点。作为一个长期目标，我们还将重点了解决定每个复合体底物专一性的分子碱基。为此，我们将对必需复合体的羧基转移酶亚基(2)进行基于结构的突变，然后对突变的酶亚基进行生化鉴定。公共卫生相关性：结核分枝杆菌导致的人类死亡比任何其他单一传染病都多，每年估计有800万新的结核病病例和200万人死亡。鉴于目前出现多药耐药结核病(MDR-TB)的背景，结核病治疗正在进入一个新的、具有挑战性的时代，有效的控制需要确定新的药物和新的药物靶点。在这个项目中，我们提出了两个结核分枝杆菌酰基辅酶A羧化酶(ACCase)复合体的特征，该复合体参与了真菌酸的生物合成，真菌酸是细菌细胞被膜中存在的复杂脂类，对于病原体的生存和致病性是必不可少的。我们希望这些酶复合体的生化、生理和结构表征将有助于我们实现该项目更长远的目标，即鉴定ACCase抑制剂，最终可能开发成新的抗分枝杆菌药物。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) in humans, is responsible for approximately three million deaths worldwide every year and estimated to infect one-third of the world's population. The re-emergence of this disease, caused by the lack of drug compliance, the appearance of multiple-drug-resistant (MDR) strains, and the AIDS epidemic, urge the development of new drugs with activity against MDR strains that could reduce the duration and complexity of current therapies. Acyl-CoA carboxylases (ACCase) have crucial roles in fatty acid metabolism in most living organisms and have been proposed as a good target for the development of a new class of antibacterial agents. In M. tuberculosis, six putative ACCases are predicted from the genome sequence and two of them, ACCase4 and ACCase6, are proposed to be involved in mycolic acid biosynthesis. This complex lipid is one of the main components of the M. tuberculosis cell envelope and has been associated with the survival and pathogenicity of this bacterium. The objective of the proposed research is to characterize ACCases4 and 6 from M. tuberculosis at the genetic, biochemical, and structural levels and understand, by using M. smegmatis as a working model, their physiological role and relevance in mycolic acid biosynthesis and in the survival of these microorganisms. Additionally, we will characterize in vivo and in vitro, putative ACCase inhibitors identified by in silico docking of chemical libraries, and use them to validate these enzyme complexes as target for the future development of antimycobacterial drugs. As a long term goal we will also put emphasis in understanding the molecular bases that determine the substrate specificity of each complex. For this, we will carry on structure based mutagenesis of the carboxyltransferase subunits (2) of the essential complex followed by the biochemical characterization of the mutated enzyme subunits. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis causes more human deaths that any other single infectious organism with an estimated eight million new tuberculosis cases and two million fatalities each year. Given the current backdrop of emerging multi-drug resistant tuberculosis (MDR-TB), TB treatment is entering a new and challenging era where effective control requires the identification of new drugs and novel drug targets. In this project, we propose the characterization of two acyl-CoA carboxylase (ACCase) complexes of M. tuberculosis involved in the biosynthesis of mycolic acids, complex lipids present in the bacterial cell envelope which are essential for the pathogen survival and pathogenicity. We expect that the biochemical, physiological and structural characterization of these enzyme complexes will help us to achieve a more long-term goal of this project, the identification of ACCase inhibitors that could eventually be developed into new antimycobacterial drugs.