The bacterial fatty acid (FAS-II) biosynthetic pathway is an attractive target for the design of antimicrobial agents due to structural and mechanistic differences between eukaryotic and prokaryotic biosynthetic enzymes.  We have recently reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against select pathogens [Hevener et al. (2012) J. Med Chem. 55, 268-79]. We have also solved the crystal structure of the FabI enzyme in complex with the NADH cofactor and with our most active benzimidazole [Mehboob et al. (2012) J. Med Chem. 55, 5933–5941], demonstrating that the benzimidazole compounds bind to FabI in a unique conformation that is distinct from the binding motif of other known FabI inhibitors.  Inhibition kinetics demonstrate that the compounds are uncompetitive with respect to the cofactor NADH and competitive with respect to the substrate - a novel inhibitory mechanism that is unique among known FabI inhibitors. More recently, we have shown that the compounds also rapidly depolarize bacterial membranes, with subsequent slower bacterial killing as compounds diffuse into the bacterial cytoplasm.  FabI overexpression systems demonstrate that bacterial killing results from FabI inhibition. This information has been used in several cycles of structure-based design, resulting in inhibitors with low µg/mL activity against multiple bacterial pathogens, including both wild type and methicillin-resistant S. aureus, and that exhibit good microsomal stability and low cytotoxicity.  This work thus reveals a new approach for the design of FAS-II active antibacterial compounds. This research was supported in part by US  NIH grant U01 AI077949.