ASSAY FOR ANTIBACTERIALS AFFECTING PPGPP DEGRADASE

The proposed research is aimed at discovering new antibacterial agents. New antibacterials are needed since bacteria have acquired resistances to most classes of currently approved antibiotics. We describe a screening system comprising a high-throughput primary assay and a secondary assay that can confirm the mechanism of action of a test compound. The high- throughput screen will identify compounds affecting a specific bacterial enzyme, ppGpp degradase. This enzyme is a crucial component of a bacterial signaling system that modulates gene expression. ppGpp degradase is not found in mammalian cells and is therefore a selective target for antibacterial activity. The proposed work will assess the utility of the screening system by screening 25,000 diverse compounds from the RiboGene compound library. Positives in the primary assay will be tested in the secondary confirmatory assay. In addition, positive compounds will be tested for antibacterial activity against a variety of bacterial species, and for cytotoxicity against mammalian cell lines. The results of these tests will be used to assess the feasibility of using the screening system in a very large scale screening for the discovery of new antibacterial agents.