(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.
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