Structural and Biochemical Studies of LpxC Inhibition

性状（由申请方提供）：脂质A（内毒素）是一种基于葡糖胺的糖脂，构成革兰氏阴性菌外膜的外层单层;也是脂多糖的活性成分，可导致危及生命的革兰氏阴性菌感染性休克。脂质A生物合成是几乎所有革兰氏阴性生物体中保守的重要途径。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）催化。由于LpxC是脂质A生物合成中的必需酶，并且与任何已知的哺乳动物蛋白不共享序列或结构同源性，因此它是用于设计新型抗生素的极好靶标。事实上，已经发现了几种有效的LpxC抑制剂，它们显示出不同程度的抗生素活性。最近发现的一些化合物也显示出时间依赖性LpxC抑制，这是抗生素非常需要的特性，因为酶/抑制剂复合物的半衰期长。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。大肠杆菌LpxC的许多有效抑制剂对不同的LpxC酶相对无活性，特别是来自铜绿假单胞菌的LpxC酶，铜绿假单胞菌是囊性纤维化患者死亡的主要原因。CHIR-090是迄今为止发现的最有效的LpxC抑制剂，对多重耐药革兰氏阴性病原体如醋酸钙不动杆菌和洋葱伯克霍尔德氏菌无效。这种不寻常的抑制剂特异性和缺乏各种LpxC/抑制剂复合物的结构信息一起严重阻碍了现有LpxC抑制剂的进一步优化。该提案的总体目标是（1）了解LpxC潜在抑制剂特异性和时间依赖性抑制的基本未知分子特征，以及（2）利用这些信息改善下一代LpxC靶向抗生素的效力和抑制谱。这一目标将通过对与代表性LpxC抑制剂复合的不同LpxC直向同源物的详细结构和生化研究，以及基于结构见解的新型化合物的设计、合成和评价来实现。对多重耐药革兰氏阴性病原体（包括对所有临床可用抗生素具有耐药性的假单胞菌属或不动杆菌属菌株）缺乏有效治疗，突出了对具有新作用机制的抗生素的迫切需求。我们提出的LpxC的结构和生化研究，在脂质A的生物合成和革兰氏阴性菌的一种新的抗生素靶标的必需酶，将揭示潜在的抑制剂特异性和时间依赖性抑制的分子基础。我们的研究已经受益，并将继续促进针对广谱革兰氏阴性病原体的有效LpxC靶向抗生素的开发。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE 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.