The increasing occurrence of pathogenic bacteria that are resistant to historically successful antibiotics has driven the need to develop new classes of drugs that have a unique mechanism of action. Gram-negative pathogens pose a serious health threat as they can be the causative agents of life-threatening conditions such as septicemia (Escherichia coli), meningitis (Haemophilus influenzae) or pyrogenic infections in burn patients and diabetics (Pseudomonas aeruginosa). 3-deoxy-D-manno-octulosonic-8-phosphate synthase (KDO8PS; EC 4.1.2.16) has been demonstrated to be an essential enzyme for gram-negative pathogen viability as it carries out a key step in the biosynthesis of the outer membrane lipopolysaccharide component, namely the condenstaion of phosphoenolpyruvate and arabinose-5-phosphate to form 3-deoxy-D-mann-octulosonic-8-phosphate. We propose to identify inhibitors of KDO8PS that can be further developed into antibiotics for use in treating infections by gram-negative pathogens. The specific aims are to clone and express the gene for KDO8PS from E. coli and P. aeruginosa, purify the enzyme to homogeneity, and develop assays for high throughput screening and compound characterization that can be used to identify and develop inhibitors of KDO8PS as potential antibiotics.
