A Fluorescence Polarization Assay for Fatty Acid Adenylating Enzymes

描述（由申请人提供）：结核分枝杆菌（Mtb）是结核病（TB）的病原，是导致细菌性传染病死亡的主要原因。结核分枝杆菌的细胞包膜提供了一个渗透性屏障，保护细菌免受环境胁迫，提供对化疗药物的内在抗性，在结核病的持久性中起关键作用，并参与分枝杆菌的发病机制。分枝杆菌细胞壁的显著特征是存在一层由各种各样的外来脂质组成的外蜡质。脂质生物合成是必不可少的，脂质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34种脂肪酸腺苷化酶FadD's参与脂质代谢。相比之下，大肠杆菌只编码一个fadD。fadD的非凡功能冗余确实令人惊讶，并清楚地说明了脂质代谢的重要性。基于序列测序和功能鉴定，分枝杆菌FadD被分为2类腺苷化酶：1)参与脂质生物合成的长链脂肪酰基- amp连接酶（FAALs）和2)参与脂质分解代谢的酰基-辅酶a合成酶（ACSs）。针对每一类FadD的特异性小分子抑制剂或单个FadD的选择性抑制剂的鉴定有望帮助破译FadD在脂质代谢中的功能作用，并可能进一步导致新型抗结核药物的开发。为了实现这一目标，我们建议开发一种适用于高通量筛选的荧光偏振法来鉴定小分子FadD抑制剂。我们的长期目标是了解结核分枝杆菌基因组编码的34个fadD的功能作用，以及它们如何促进分枝杆菌的发病机制。本申请的目的是开发和验证一种适合高通量筛选FadD32 （FadD32是霉菌酸生物合成所需的重要基因，是FAAL类FadD的代表成员）和FadD19 （FadD19是分枝杆菌持久性所需的胆固醇分解代谢基因，是FadD的酰基辅酶a合成酶类的代表成员）的荧光极化测定法。在HTS试验中鉴定的化合物将通过已建立的焦磷酸盐交换放射性试验作为二次筛选进行验证。随后针对一组FadD的反筛选将获得鉴定化合物的选择性，这可能是询问分枝杆菌脂质代谢的有用工具。

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.