BIOSYNTHESIS AND FUNCTION OF LIPOPOLYSACCHARIDES

描述：(来自申请书)外膜的外叶 革兰氏阴性细菌的表面覆盖着一种引人注目的大分子 被称为脂多糖(LPS)的糖脂。在大肠杆菌中， 脂蛋白A锚点是氨基葡萄糖的六酰化二糖， 在1‘和4’位含磷的。所需的最低内毒素 大肠杆菌的生长含有5个脂酰链和2个额外的 加糖。脂类A的生物合成具有很好的特性。遗传或 对催化第一种酶的任何一种酶的药物抑制 在大肠杆菌中，该途径的七个步骤会导致细胞死亡。新兴基因组学 不同细菌的序列表明这些酶存在于 几乎所有的革兰氏阴性生物。因此，类脂A是一种有趣的 成为设计新型抗菌剂的目标。 脂蛋白A(又称内毒素)是脂多糖的活性成分，可 刺激免疫细胞。在严重的革兰氏阴性感染期间，脂质 从细菌中排出的部分内毒素可导致过度激活 巨噬细胞和内皮细胞。由此导致的系统性生产过剩 炎症介质，如肿瘤坏死因子，白介素1和白介素6，损害 微血管系统。对内毒素的完全反应导致革兰氏阴性 感染性休克和死亡。一种令人兴奋的新方法解决了这个问题 发现某些类脂A分子，包括 一些类脂A前体是内毒素拮抗剂。卫材公司 最近报道了化合物E5531，一种有效的合成拮抗剂 在阻止I期人类体内的内毒素挑战方面非常有效 审判。还没有其他分子被描述为能阻止内毒素 行动如此彻底。E5531目前处于第二阶段临床试验。 在此之前，申请者阐明了关键的酶步骤和 脂类A在大肠杆菌中组装的遗传学研究。一些酶现在能适应于 结构生物学，为脂肪/蛋白质识别提供了新的见解。 在即将到来的赠款期间，具体目标是：i)确定 脂A生物合成第一酶(LpxA)的活性部位；II) 脂类A生物合成调控机制的测定，包括 这些是由抑制第二个(已承诺的)酶触发的 途径(LpxC)和冷休克；iii)鉴定新的酶和 参与脂类A生物合成的基因：IV)生化和遗传学 A类脂转运的特征，特别是MSBA的作用；和V) 含反应基团的新型放射性类脂A类似物的制备 用于探测脂质A结合部位I蛋白。

DESCRIPTION: (From the application) The outer leaflet of the out membrane of Gram-negative bacteria is covered with a remarkable, macromolecular glycolipid known as lipopolysaccharide (LPS). In Escherichia coli, the lipid A anchor of LPS is a hexaacylated disaccharide of glucosamine, bearing phosphate at the 1 and 4' positions. The minimal LPS required for growth of E. coli contains five acyl chains on lipid A nd two additional sugars. The biosynthesis of lipid A is well characterized. Genetic or pharmacological inhibition of any one of the enzymes catalyzing the first seven steps of the pathway in E. coli causes cell death. Emerging genomic sequences of diverse bacteria indicate that these enzymes are present in almost all Gram-negative organisms. Lipid A is, therefore, an interesting target for deigning new antibacterial agents. Lipid A (also known as endotoxin is the active component of LPS that stimulates immune cells. During severe Gram-negative infections the lipid A moiety of LPS shed from bacteria can cause excessive activation of macrophages and endothelial cells. The resulting systemic over-production of inflammatory mediators, such as TNF, IL-1 and IL-6, damages the microvasculature. A full response to endotoxin leads to Gram-negative septic shock and death. An exciting new approach to this problem has emerged with the discovery that certain lipid A-like molecules, including some lipid A-like precursors, are endotoxin antagonists. The Eisai company has recently reported compound E5531, a potent synthetic antagonists that is very effective in blocking an endotoxin challenge in phase I human trials. No other molecules has ever been described that blocks endotoxin action so completely. E5531 is now in phase II clinical trials. Previously, the applicant elucidated the key enzymatic steps and the genetics of lipid A assembly in E. coli. Some enzymes are now amenable to structural biology, providing new insights into lipid/protein recognition. In the coming grant period, the specific aims are: I) Identification of the active site of the first enzyme (LpxA) of lipid A biosynthesis; II) determination of regulatory mechanisms of lipid A biosynthesis, including those triggered by inhibition of the second (committed) enzyme of the pathway (LpxC), and cold shock; III) identification of novel enzymes and genes involved in lipid A biosynthesis: IV) biochemical and genetic characterization of lipid A transport, especially the role of MsbA; and V) preparation of new radioactive lipid A analogs containing reactive groups for probing lipid A binding sites i proteins.