Structural and Biochemical Studies of LpxC Inhibition

说明(申请人提供)：脂类A(内毒素)是一种以氨基葡萄糖为基础的糖脂，构成革兰氏阴性细菌外膜的外单分子层；它也是脂多糖的活性成分，可导致危及生命的革兰氏阴性败血症休克。脂质A的生物合成是几乎所有革兰氏阴性生物都保守的一条重要途径。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)催化的。由于LpxC是脂质A生物合成的关键酶，与任何已知的哺乳动物蛋白质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极佳靶标。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还显示出时间依赖性的LpxC抑制，这一特性对于抗生素来说是非常可取的，因为酶/抑制剂复合体的半衰期很长。不同的LpxC同源基因之间可能存在很大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酶相对无效，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的主要死亡原因。CHIR-090是迄今为止发现的最有效的LpxC抑制剂，对耐多药的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专一性和缺乏关于各种LpxC/抑制剂复合体的结构信息，共同严重阻碍了现有LpxC抑制剂的进一步优化。这项建议的总体目标是(1)了解LpxC潜在的抑制剂特异性和时间依赖抑制的基本未知的分子特征，(2)利用这些信息来提高下一代LpxC靶向抗生素的抑制效力和抑制谱。这一目标将通过对具有代表性的LpxC抑制剂复合体中不同的LpxC同系物进行详细的结构和生化研究，以及基于结构洞察力设计、合成和评估新化合物来实现。公共卫生相关性缺乏对多重耐药革兰氏阴性病原体的有效治疗，包括对所有临床上可用的抗生素都具有耐药性的假单胞菌或不动杆菌菌株，这突显了对具有新作用机制的抗生素的迫切需要。我们对LpxC的结构和生化研究将揭示抑制剂专一性和时间依赖性抑制的分子基础。LpxC是脂质A生物合成的关键酶，也是革兰氏阴性菌的新抗生素靶点。我们的研究已经受益，并将继续促进有效的LpxC靶向抗生素的开发，以对抗广谱的革兰氏阴性病原体。

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.