There is a worldwide crisis in management of drug-resistant bacterial infections. In particular Gram-positive (Gram+) bacteria such as Staphylococcus aureus, Enterococcus fecalis and Enterococcus fecium are increasingly resistant to traditional antibiotics. This project focuses on inhibitors of a newly described DNA polymerase in Gram+ bacteria, pol IIIE, and builds upon previous results obtained from our work with inhibitors of pol IIIC, the 6-anilinouracils (AUs), in antibacterial drug discovery. We have identified potent lead inhibitors of pol IIIE from Gram+ bacteria - N2,7-disubstituted guanines - that act as competitive, active site-directed inhibitors of pol IIIE. Based on these observations we will pursue antibiotic drug discovery through these specific aims: 1. to use parallel synthesis methods to synthesize N2-substituted guanines, 7-substituted-N2-substituted guanines, and N2,7-disubstituted guanines; 2. to synthesize isosteres of the most potent N2,7-disubstituted guanines - 3-deaza, 8-aza and 3-deaza-8-aza guanines predicted to be highly potent pol IIIE inhibitors; 3. to assay compounds for their capacity to inhibit pol IIIE and pol IIIC isolated from the Gram+ bacteria B. subtilis, S. aureus, and E. fecalis, and from the Gram- bacterium E. coli; 4. to assay compounds against Gram+ and Gram- bacteria, and for cytotoxicity against human cells; 5. to design, based on the results of aims 1-3, one or more pol IIIE inhibitors suitable for pharmacokinetic and efficacy studies in mouse infection models during phase II of the project. Potent inhibition of Gram+ pol IIIE will lead to candidate antibacterials with reduced incidence of resistance. In addition, activity against the Gram- pol IIIE from E. coli by our lead inhibitor indicates the possibility of designing a truly broad spectrum antibacterial compound derived from this scaffold. PROPOSED COMMERCIAL APPLICATION: A new antibiotic drug capable of curing infections caused by drug-resistant bacteria can have a significant market. The compounds developed in this project have potential utility against infections caused by Gram+ bacteria, and, by virtue of being active against more than one enzymatic target, will have a low tendency to develop resistance. Truly broad spectrum drugs may be developed if such compounds inhibit targets in both Gram+ and Gram- bacteria.