Exploring the Antibacterial Activity and Cellular Fates of Enterobactin–Drug Conjugates That Target Gram-Negative Bacterial Pathogens

Conspectus Siderophores are secondary metabolites utilized by bacteria to acquire iron (Fe), an essential transition metal nutrient. Fe levels in the host environment are tightly regulated and can be further restricted to starve invading bacterial pathogens in a host-defense process known as nutritional immunity. To survive and colonize the Fe-limited host environment, bacteria produce siderophores and express cognate siderophore transport machinery. These active transport pathways present an opportunity for selective and efficient drug delivery into bacterial cells, motivating decades of research on synthetic siderophore–antibiotic conjugates (SACs) as a Trojan-horse strategy for the development of targeted antibiotics. Enterobactin (Ent) is a triscatecholate siderophore produced and utilized by many Gram-negative bacteria, including all Escherichia coli and Salmonella species. Within these species, pathogenic strains cause a variety of human diseases including urinary tract infections, gastroenteritis, and sepsis. Infections caused by these Gram-negative pathogens can be difficult to treat because of the impermeability of the outer membrane (OM). This impermeability can be overcome by utilizing siderophores as drug delivery vectors for targeting Gram-negative pathogens. Ent is a promising delivery vector because it undergoes active transport across the OM mediated by the Ent uptake machinery after scavenging Fe(III) from the extracellular environment. Despite the well-elucidated chemistry and biology of Ent, its use for SAC development was hampered by the lack of an appropriate functional group for cargo attachment. Our laboratory addressed this need by designing and synthesizing monofunctionalized Ent scaffolds. Over the past decade, we have used these scaffolds to explore Ent-based SACs with a variety of drug warheads, including β-lactam and fluoroquinolone antibiotics, and Pt(IV) prodrugs. Investigations of the antibacterial activities of these conjugates and their cellular fates have informed our design principles and revealed approaches to achieving enhanced antibacterial potency and pathogen-targeted activity. Collectively, our studies of Ent–drug conjugates have provided discoveries, understanding, and invaluable insights for future design and evaluation of SACs. In this Account, we present the story of our work on Ent–drug conjugates that began about ten years ago with the development of monofunctionalized Ent scaffolds and the design and synthesis of various conjugates based on these scaffolds. We describe the antibacterial activity profiles and uptake pathways of Ent–drug conjugates harboring traditional antibiotics and repurposed platinum anticancer agents as well as studies that address cellular targets and fates. Finally, we discuss other applications of monofunctionalized Ent scaffolds, including a siderophore-based immunization strategy. We intend for this Account to inspire further investigations into the fundamental understanding and translational applications of siderophores and siderophore–drug conjugates.