Next-Generation Antibiotics

The outer membrane of Pseudomonas aeuruginosa is more impermeable than that of most other Gram-negative bacteria, contributing significantly to antibiotic resistance. A major component of the outer membrane is lipopolysaccharide (LPS), and understanding its synthesis suggests new targets for antibiotics. WaaP is a kinase responsible for phosphorylating sugar residues in the LPS core region and conferring considerable stability via ionically bonded divalent cations. Mutations of waaP in Pseudomonas are fatal, suggesting that inhibitors of WaaP will act as lethal antibiotics. WaaP in Pseudomonas has also been shown to have protein kinase activity in addition to sugar kinase activity. The work proposed is directed toward developing an assay of WaaP requiring minimal mechanical steps and with well-characterized biochemical kinetics in order to design an HTS assay with the greatest probability of detecting small molecule inhibitors of WaaP. We also propose to examine known mammalian kinase inhibitors in WaaP to optimize the assay for detection of inhibitors in HTS, and to provide an opportunity to compare the mechanism of action of known mammalian kinase inhibitors with new inhibitors of WaaP. This should also allow us to choose compounds for development with the least probability of inhibiting mammalian protein kinases.