The rapid emergence of antimicrobial resistance presents a significant challenge for treatment of bacterial infections. Carbapenem-resistant Enterobacteriaceae (CRE), Acinetobacter baumannii, and Pseudomonas aeruginosa are of particular concern. We are clearly in need of several new Gram-negative agents that are unique in terms of liabilities and antimicrobial class and which can diversify our antimicrobial development portfolio. This multi-PI proposal investigates natural products called streptothricins, which contain three moieties: streptolidine, a gulosamine sugar, and a single ß-lysine or poly-ß-lysine chain of varying length. Streptothricins were identified over 70 years ago and inhibit protein translation with extensive protein miscoding. In preliminary experiments, we determined that streptothricins (the natural product mixture is also called nourseothricin) are broadly active against multidrug-resistant Gram-negative pathogens. In particular, for streptothricin-F, which has a single ß-lysine moiety, we identified compelling activity in vitro and in vivo. However, several streptothricin acetyl transferases, found in low frequency in Gram-negative pathogens, confer streptothricin resistance by acetylation of the ß-amine of the ß-lysine residue. These observations led to our hypothesis that antibacterial activity can be separated from toxicities and at the same time these antibiotic resistance elements blocked through derivatization/replacement of the ß-lysine moiety and other constituents. Therefore, the goals of this proposal are to use an efficient, diversity- oriented, medicinal chemistry synthesis of streptothricin analogues to perform hypothesis-driven structure- activity relationship studies to optimize therapeutic properties of this scaffold. In particular, we propose to functionally profile each streptothricin analogue to determine potency against problematic Gram-negative pathogens, selectivity for prokaryotic ribosomes, toxicity, and metabolic stability. Prioritized analogues will be tested in a mouse model for toxicity, drug clearance, and therapeutic efficacy. Furthermore, prioritized analogues will be investigated in cryo-EM based-structural and auto-docking studies to understand how these molecular variants differentially bind to the A. baumannii 70S ribosome. Structural insights from these studies will be used to provide iterative feedback to optimize design of analogues during the course of the proposed work. Taken together, the experiments in the aims of the proposal will address the lack of systematic exploration in the streptothricin literature and identify molecular constituents that are amenable to productive modification to enhance properties of this scaffold as a future therapeutic.