A Fluorescence Polarization Assay for Fatty Acid Adenylating Enzymes

描述(申请人提供)：结核分枝杆菌(Mtb)，结核病(TB)的病原体，是细菌感染性疾病死亡的主要原因。结核分枝杆菌的细胞膜提供了一种通透性屏障，使其免受环境压力的影响，对化疗药物具有内在的耐药性，在结核杆菌的持久性中起着关键作用，并参与了分枝杆菌的发病机制。分枝杆菌细胞壁的显著特征是外层有一层蜡状的外皮，由各种外来脂类组成。虽然脂类生物合成是必不可少的，但脂类分解代谢在分枝杆菌的生理和潜伏期中也起着关键作用。结核分枝杆菌编码了一种令人惊讶的34种脂肪酸腺化酶，FADD参与了脂肪代谢。相比之下，大肠杆菌编码一个单一的FADD。FADD的非凡功能冗余真的令人惊叹，清楚地说明了脂代谢的重要性。根据序列比对和功能鉴定，分枝杆菌FADD可分为两类：1)与脂质生物合成有关的长链脂肪酰基-AMP连接酶(FAALs)；2)与脂质分解代谢有关的酰基-辅酶A合成酶(ACSS)。识别针对每一类FADD的特定小分子抑制剂或针对单个FADD的选择性抑制剂有望帮助破译FADD在脂代谢中所起的功能作用，并可能进一步导致新类型抗结核药物的开发。为了实现这一目标，我们建议发展一种适合高通量筛选的荧光偏振分析方法来鉴定小分子FADD抑制剂。我们的长期目标是了解结核分枝杆菌基因组中编码的34个FADD的功能作用以及这些作用是如何促进分枝杆菌的发病。本应用的目的是建立和验证FadD32(FadD32是霉菌酸生物合成所必需的基因，是FadD的Faal类代表成员)和FadD19(FadD19是参与胆固醇分解代谢的基因，对于分枝杆菌的持久性是必需的，也是FADD的酰基辅酶A合成酶类的代表成员)的高通量筛选的荧光偏振分析方法。HTS检测中确定的化合物将通过已建立的焦磷酸盐交换无线电检测作为二次筛选进行验证。随后针对一组FADD进行的计数器筛查将获得已鉴定化合物的选择性，这可能是询问分枝杆菌脂代谢的有用工具。 公共卫生相关性：结核分枝杆菌是结核病(TB)的病原体，是世界上由细菌病原体引起的传染病死亡的主要原因。此外，结核分枝杆菌和其他非典型分枝杆菌现在被归类为艾滋病患者的机会性感染。预计这项拟议的研究将能够识别具有新作用机制的新的抗结核药物。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), is the leading cause of bacterial infectious disease mortality. The cell envelope of M. tuberculosis provides a permeability barrier, which shields the bacterium from environmental stress, provides intrinsic resistance to chemotherapeutic agents, plays a key role in TB-persistence, and participates in mycobacterial pathogenesis. The distinguishing feature of the mycobacterial cell wall is the presence of an outer waxy coat comprised of an extraordinary variety of exotic lipids. While lipid biosynthesis is essential, lipid catabolism also plays a key role in mycobacterial physiology and latency. M. tuberculosis encodes for an astonishing 34 fatty acid adenylating enzymes FadD's involved in lipid metabolism. By contrast E. coli encodes a single fadD. The extraordinary functional redundancy of the fadD's is truly amazing and clearly illustrates the importance of lipid metabolism. Based on sequence alignment and functional characterization, the mycobacterial FadD's have been divided into 2 classes of adenylating enzymes: 1) long chain fatty acyl-AMP ligases (FAALs) involved in lipid biosynthesis and 2) acyl-CoA synthetases (ACSs) involved in lipid catabolism. The identification of specific small molecule inhibitors against each class of FadD or selective inhibitors of an individual FadD is expected to help decipher the functional role of the FadD's play in lipid metabolism and could additionally lead to the development of new class of antitubercular agents. Toward this goal we propose to develop a fluorescence polarization assay amenable to high throughput screening to identify small molecule FadD inhibitors. Our long-term goal is to understand the functional role of the 34 fadD's encoded in the M. tuberculosis genome and how these contribute to mycobacterial pathogenesis. The objective of this application is to develop and validate a fluorescence polarization assays amenable to high-throughput screening of FadD32 (fadD32 is an essential gene required for mycolic acid biosynthesis and a representative member of the FAAL class of FadDs) and FadD19 (fadD19 is a gene implicated in cholesterol catabolism that is required for mycobacterial persistence and a representative member of the acyl-CoA synthetases class of FadD's). Compounds identified in the HTS assay will be validated by an established pyrophosphate exchange radio assay as a secondary screen. Subsequent counter screening against a panel of FadD's will access the selectivity of identified compounds, which may represent useful tools to interrogate mycobacterial lipid metabolism. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis the causative agent of Tuberculosis (TB) is the leading cause of infectious disease mortality in the world by a bacterial pathogen. Additionally, M. tuberculosis and other atypical mycobacteria are now classified as opportunistic infections of AIDS patients. The proposed research is expected to enable identification of new antitubercular agents with novel mechanisms of action.