DESCRIPTION (provided by applicant): Fatty acid biosynthesis by a type II dissociated fatty acid synthase (FAS) is a fundamental and indispensable metabolic pathway in many organisms, including bacteria and parasites. The distinctions between this and the multifunctional type I FAS of higher organisms offer a selective target for the design of novel antibacterial and antiparasitic agents, needed to combat the resurgence of antibiotic-resistant bacteria and multidrug-resistant forms of the malaria-causing plasmodial parasites. In all type II FAS systems, the initial condensation step is carried out by 3-ketoacyl acyl carrier protein (ACP) synthase III (KASIII, FabH), which catalyzes the condensation of an acyl CoA substrate with malonyl ACP to generate a 3-ketoacyl ACP product. FabH also appears to play a key role in regulation of fatty acid biosynthesis and is not targeted by any current drugs, making it a particularly attractive new target for drug design. The long-term objective of our work is to understand the structural and mechanistic bases of FabH that define its physiological roles and to use this information to generate novel potent and selective inhibitors. This grant will extend our study of FabH enzymes from organisms such as Escherichia coil and Staphylococcus aureus, which initiate de novo fatty acid biosynthesis from different short chain acyl CoA substrates, and Mycobacterium tuberculosis, which uses FabH to initiate mycolate biosynthesis from long chain acyl CoA substrates. Mutational analyses and crystallography will be used to investigate the differing substrate and inhibitor specificities of these enzymes. In conjunction with ongoing crystallographic, molecular modeling and kinetic analyses, the mode of binding of 1,2-dithiole-3-one and related compounds which are potent novel active site FabH inhibitors, and a second series of inhibitors that appear to bind in the FabH phosphopantetheine binding channel, will be investigated. The information gathered from these studies will be used to design, synthesize and ultimately test inhibitors, which maximize interactions with both active site residues and those in either the acyl-binding pocket or phosphopantetheine-binding channel. Such compounds should have enhanced activity and selectivity against FabH and be powerful new lead compounds for development of novel antibacterial and antiparastitic/antimalarial drugs.