Abstract The emergence of multi-drug resistant Gram-negative pathogens (especially Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and the Enterobacteriaceae) represents the most serious challenge faced by infectious disease clinicians today. This situation highlights the urgent need for new therapeutics and innovative new therapeutic approaches to successfully treat these infections. In the initial period of this R01 project, we developed a novel small-molecule approach for the Fe2+-dependent delivery of diverse drug (or reporter) payloads. This platform has now been well validated in cells and in mouse models of malaria and cancer. In the next period of the project, we will apply and optimize this strategy to target important Gm-negative “ESKAPE” pathogens. Our hypothesis is that host-pathogen interactions resulting from competition for iron resources present multiple opportunities for intervention with Fe2+-targeted antibiotics. The various iron acquisition strategies employed by P. aeruginosa alone predict for targetable pools of labile Fe2+ in the extracellular, periplasmic, and cytoplasmic compartments, depending on disease state and site of infection. Our research plan calls for the synthesis of Fe2+-targeted antibiotic conjugates designed to passively (Aim 1) or actively (via siderophore-mediated uptake; Aim 2), transit Gm-negative membranes, and release compartment- appropriate antibiotic payloads following reaction with Fe2+. To study and verify their modes of transport and activation, we will employ non-Fe2+ reactive control conjugates, and a carefully selected panel of wild-type and mutant strains with altered permeability, efflux pump/porin expression, or siderophore/transporter expression. Susceptibility (MIC) testing will be performed under iron depleted and iron replete conditions using disease- relevant, context specific, iron sources to mimic the microenvironment of the host-pathogen interaction around iron. The most effective conjugates will be evaluated against a panel of Gm-negative clinical isolates obtained from diverse infection sites and disease states. These studies will provide new insights into the iron acquisition pathways of Gm-negative bacteria, and will inform the selection of promising leads for further study in murine infection models. Finally, to exploit the extracellular Fe2+ produced during P. aeruginosa biofilm formation, we will synthesize and characterize the first Fe2+-sensitive “smart” materials (Aim 3). Used as coatings on indwelling devices, these novel materials would detect biofilm formation in situ and deliver antibiotics exactly where and when they are needed. In summary, this project will apply the insights and chemical strategies developed during the initial R01 period to a new therapeutic area, with the aim of introducing several new innovations to the field of antibacterial therapy.