Project Summary – Project 1 - Exploiting metabolic vulnerabilities Central metabolic pathways are critical for bacterial viability and represent a rich source of potential drug targets. However, these enzymes have been under-exploited by anti-bacterial drug development programs for two basic reasons. Firstly, these complex pathways perform diverse roles under different growth states, leaving their importance during infection unclear. In addition, homologous mammalian enzymes often exist, necessitating the design of specific inhibitors. To overcome these complications and exploit this rich source of potential drug targets, we use a combination of genetics and chemical biology to identify druggable metabolic enzymes that are essential in relevant host microenvironments, and employ structural biology to aid in the development of specific inhibitors. These studies have defined target-inhibitor pairs in a broad range of metabolic pathways and regulators. For example, central carbon metabolic enzymes critical for bacterial survival in animal models, such as phophoenolpyruvate carboxykinase (PEPCK), pyruvate kinase (PK), and malate dehydrogenase, have been subjected to structure-guided inhibitor design. Similar approaches have been applied to critical components of lipid anabolic pathways, such as PKS13 and components of acyl-CoA carboxylase complexes, enzymes critical for amino acid and cofactor synthesis, and heme metabolism. These studies have identified potent and specific small molecule inhibitors, revealed novel protein complexes and elucidated the physiological functions of these enzymes in the bacterium.