SBIR-STTR Award

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.

Keywords:
ANTIBIOTICS, ANTIBIOTIC RESISTANCE, PSEUDOMONAS AERUGINOSA, KINASE INHIBITORS, HIGH-THROUGHPUT SCREENING

Finding new antibiotics to treat infections caused by Pseudomonas aeruginosa is a compelling medical need, since the inherently antibiotic resistant nature of the bacteria is being enhanced by the increasing appearance of strains resistant to all currently-marketed antibiotics. Lipopolysaccharide (LPS) is a required and constitutive component of the outer membrane of Gram-negative bacteria, including Ps. aeruginosa, Escherichia coli, Salmonella and Shigella species, which contributes significantly to antibiotic resistance. WaaP is a kinase encoded by these organisms and is required for complete synthesis of LPS. Knock-out mutations in the waaP gene are lethal in Ps. aeruginosa thus making inhibition of WaaP a useful target for the development of new antibiotics. The goal of the proposed project is to find small molecule inhibitors of WaaP that will be developed into a new class of narrow-spectrum antibiotics especially targeted to Ps. aeruginosa. The research proposed will support high-throughput screening of a 100,000+ compound library and numerous follow-up assays in order to identify compounds with pharmaceutical properties suitable to serve as templates for antibiotic development. The proposal also describes intensive medicinal chemistry efforts and biological and early toxicological evaluations of newly synthesized drug candidates.

Keywords:
Antibiotics, Antibiotic Resistance, Pseudomonas Aeruginosa, Kinase Inhibitors, High-Throughput Screening