Structural and Biochemical Studies of LpxC Inhibition

描述（由申请人提供）：脂质A（内毒素）是一种以葡萄糖胺为基础的糖脂，构成革兰氏阴性菌外膜的外单层；它也是导致危及生命的革兰氏阴性脓毒性休克的脂多糖的活性成分。脂质A生物合成在几乎所有革兰氏阴性菌中都是一个重要的保守途径。脂质A生物合成的关键步骤是由UDP-3-O-（酰基）- n -乙酰氨基葡萄糖脱乙酰酶（LpxC）催化。由于LpxC是脂质A生物合成中必需的酶，并且与任何已知的哺乳动物蛋白都没有相同的序列或结构同源性，因此它是设计新型抗生素的理想靶点。事实上，已经发现了几种有效的LpxC抑制剂，它们表现出不同程度的抗生素活性。最近发现的一些化合物还显示出时间依赖性的LpxC抑制作用，由于酶/抑制剂复合物的半衰期很长，这种特性对于抗生素来说是非常理想的。不同的LpxC同源物之间可能存在显著程度的局部结构差异。许多有效的大肠杆菌LpxC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，这是囊性纤维化患者死亡的主要原因。chr -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无效，如钙酸不动杆菌和洋葱伯克霍尔德菌。这种不寻常的抑制剂特异性以及各种LpxC/抑制剂复合物结构信息的缺乏严重阻碍了现有LpxC抑制剂的进一步优化。本提案的总体目标是：(1)了解LpxC潜在抑制剂特异性和时间依赖性抑制的大部分未知分子特征；(2)利用这些信息来提高下一代LpxC靶向抗生素的效力和抑制谱。这一目标将通过对具有代表性的LpxC抑制剂的不同LpxC同源物的详细结构和生化研究，以及基于结构见解的新化合物的设计、合成和评估来实现。由于缺乏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耐药的假单胞菌或不动杆菌）的有效治疗，因此迫切需要具有新型作用机制的抗生素。LpxC是脂质A生物合成的必需酶，也是革兰氏阴性菌的新型抗生素靶点，我们提出的LpxC的结构和生化研究将揭示抑制剂特异性和时间依赖性抑制的分子基础。我们的研究已经并将继续促进针对广谱革兰氏阴性病原体的高效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.