Structural and Biochemical Studies of LpxC Inhibition

项目总结/摘要 脂质A（内毒素）是一种基于葡糖胺的糖脂，其构成外膜的外单层。 革兰氏阴性菌的膜;它也是脂多糖的活性成分，导致生命- 有革兰氏阴性菌感染性休克的危险脂质A的生物合成是一个重要的途径，几乎所有的保守 革兰氏阴性菌。脂质A生物合成的关键步骤由UDP-3-O-（酰基）-N- 乙酰葡糖胺脱乙酰酶（LpxC）。因为LpxC是脂质A生物合成中的必需酶， 与任何已知的哺乳动物蛋白质不共享序列或结构同源性，它是免疫组织化学的极好靶点。 设计新型抗生素。事实上，已经发现了几种有效的LpxC抑制剂，其表现出各种不同的特性。 抗生素活性的程度。最近发现的一些化合物也显示出时间依赖性LpxC 抑制，由于酶/抑制剂的半衰期长，这是抗生素高度期望的特性 复杂. 不同的LpxC直系同源物之间可能存在显著程度的局部结构变异。许多 大肠杆菌LpxC的有效抑制剂对不同的LpxC酶相对无活性，特别是 铜绿假单胞菌是导致囊性纤维化患者死亡的主要原因。CHIR-090，最 迄今为止发现的一种有效的LpxC抑制剂对多重耐药革兰氏阴性病原体无效 如醋酸钙不动杆菌和洋葱伯克霍尔德氏菌。这种不寻常的抑制剂特异性和缺乏 关于各种LpxC/抑制剂复合物的结构信息一起严重阻碍了LpxC/抑制剂复合物的进一步优化。 现有的LpxC抑制剂 该提案的总体目标是（1）了解LpxC的大部分未知分子特征 潜在的抑制剂特异性和时间依赖性抑制，以及（2）利用该信息来改善这两者 下一代LpxC靶向抗生素的效力和抑制谱。这一目标将 通过对不同LpxC直系同源物的详细结构和生物化学研究， 代表性的LpxC抑制剂，并通过设计、合成和评价基于 结构性的洞察力。项目叙述（公共卫生相关性声明） 缺乏对多重耐药革兰氏阴性病原体的有效治疗， 假单胞菌属或不动杆菌属对所有临床可用的抗生素都有耐药性， 需要具有新作用机制的抗生素。 我们提出的LpxC的结构和生物化学研究，在脂质A的生物合成的一个重要酶， 革兰氏阴性菌的新型抗生素靶点，将揭示抑制剂的分子基础 特异性和时间依赖性抑制。我们的研究已经使我们受益，并将继续促进 开发针对广谱革兰氏阴性病原体的有效LpxC靶向抗生素。

Project Summary/Abstract Lipid A (endotoxin) is a glucosamine-based saccharolipid that constitutes the outer monolayer of the outer membrane of Gram-negative bacteria; it is also the active component of lipopolysaccharide that causes life- threatening Gram-negative septic shock. Lipid A biosynthesis is an essential pathway conserved in virtually all Gram-negative organisms. The committed step of lipid A biosynthesis is catalyzed by UDP-3-O-(acyl)-N- acetylglucosamine deacetylase (LpxC). Because LpxC is an essential enzyme in lipid A biosynthesis and does not share sequence or structural homology with any known mammalian protein, it is an excellent target for the design of novel antibiotics. Indeed, several potent LpxC inhibitors have been discovered that display various degrees of antibiotic activity. Some of the recently discovered compounds also show time-dependent LpxC inhibition, a property that is highly desirable for an antibiotic because of the long half-life of the enzyme/inhibitor complex. A significant degree of local structural variation is likely to exist among different LpxC orthologs. Many of the potent inhibitors of Escherichia coli LpxC are relatively inactive against divergent LpxC enzymes, especially that from Pseudomonas aeruginosa, the leading cause of death in cystic fibrosis patients. CHIR-090, the most potent LpxC inhibitor discovered to date, is ineffective against multidrug-resistant Gram-negative pathogens such as Acinetobacter calcoaceticus and Burkholderia cepacia. This unusual inhibitor specificity and the lack of structural information on various LpxC/inhibitor complexes together severely hinder further optimization of existing LpxC inhibitors. The overall goal of this proposal is (1) to understand the largely unknown molecular features of LpxC underlying inhibitor specificity and time-dependent inhibition and (2) to utilize this information to improve both the potency and spectrum of inhibition for the next generation of LpxC-targeting antibiotics. This goal will be achieved by detailed structural and biochemical studies of divergent LpxC orthologs in complex with representative LpxC inhibitors, and by design, synthesis and evaluation of novel compounds based on structural insights. Project Narrative (Public Health Relevance Statement) The lack of effective treatment for multidrug-resistant Gram-negative pathogens, including strains of Pseudomonas or Acinetobacter that are resistant to all clinically available antibiotics, underscores the pressing need for antibiotics with novel mechanisms of action. Our proposed structural and biochemical studies of LpxC, an essential enzyme in lipid A biosynthesis and a novel antibiotic target of Gram-negative bacteria, will reveal the molecular basis underlying inhibitor specificity and time-dependent inhibition. Our studies have already benefited and will continue to facilitate the development of potent LpxC-targeting antibiotics against a broad spectrum of Gram-negative pathogens.