The prokaryotic fatty acid biosynthetic machinery constitutes an essential and potentially rich source of novel and unexploited targets for antibacterial drug development. SmithKline Beecham Pharmaceuticals (SB) has developed a major center of excellence in this research area and we propose to leverage this expertise in this project to insure development of antibiotics for diseases that might otherwise not benefit from such a program such as tuberculosis. Thiolactomycin (TLM) has been used extensively as a tool in the biochemical characterisation of beta-ketoacyl-ACP synthases (KAS) but little thought has been given in recent years to it's possible utility as the basis of a drug discovery effort. It has many features desirable in a drug including good toxicological profiles and demonstrated in vivo efficacy in animal models of bacterial infection. A key aspect of this proposal will be the application of the considerable microbial biochemical and medicinal chemistry expertise at SB and in Dr. Clif Barry's lab in the intramural research program of NIH to investigate the optimization of thiolactomycin via synthesis of a focussed library of TLM analogs using combinatorial chemical approaches. To facilitate the evaluation of TLM analogs, and the identification of other promising lead molecules through high- throughput screening, KAS and associated FAS II enzymes will be cloned, overexpressed, and purified from several key pathogens, including Mycobacterium tuberculosis, Staphylococcus aureus and Enterococcus faecalis, and assays will be configured for high throughput screening (HTS). HTS will be run using these KAS assays against SB's extensive in-house drug discovery library allowing SB to develop a portfolio of compounds which are suitable for evaluation and optimization against these target enzymes. Furthermore, state-of-the-art mechanistic enzymology, structural biology, in vitro and in vivo testing (including DMPK/PD) will be applied to TLM and more active analogs of TLM to maximize opportunities to convert promising active leads into genuine drug candidates. The R and D activities described above hold out the very attractive and attainable promise of developing lead compounds suitable for development as novel antibacterial agents against multi-drug resistant S.aureus (MRSA), vancomycin- resistant enterococci (VRE) and multi-drug resistant M.tuberculosis (MDRTB). By focussing on a target central to bacterial metabolism that differs fundamentally from the corresponding eukaryotic enzyme system, these studies will allow us to develop a novel compound series with broad-spectrum potential, which should be insensitive to current major resistance mechanisms, wherein individual chemical entities may show exquisite specificity for individual pathogens.