Recently, WHO and CDC designated Carbapenem-resistant Enterobacteriaceae (CRE) a Priority 1 ‘critical superbug’ and an ‘Urgent Threat’, and warned that new treatments for superbugs, which kill nearly 50,000 Americans and Europeans a year, are unlikely to be developed in time if left to market forces alone. Few therapeutic options are left to treat CRE, and the fear of ‘pan-resistant’ CRE has emerged. Currently, most CRE infections occur in a hospital setting, but the potential spread of CRE in the community also exists. Entirely new agents with novel mechanisms of action languish; completely novel antibacterials with a new mechanism of action and lacking clinical cross-resistance to existing drug classes are urgently needed. Our proposal aims to develop a mechanistically novel, IV and PO administered agent to treat uncomplicated and complicated UTI cases caused by antibiotic susceptible and multidrug-resistant Enterobacteriaceae, including extended spectrum beta-lactamase producers and CRE. Using an innovative overexpression-based co-culture screen in Escherichia coli (Ec), we identified four structurally distinct series of small molecule inhibitors targeting MsbA, an essential and broadly conserved Gram-negative (GN) ABC transporter responsible for lipopolysaccharide (LPS) biogenesis and construction of the Gram-negative outer membrane (OM). A prioritization of the four series through our current R21AI146541-supported program has enabled selection of a series to enter the Lead Identification (ID) phase of development. We propose the following aims to develop this series: Aim 1 (2 years) - Lead ID to select a single lead series with WT activity and efficacy for Aim 2 Lead Optimization (Op): 1.1) exploratory med chem to improve WT activity, 1.2) obtain high resolution MsbAi-MsbA X-ray co-crystal structure to guide structure-based drug design (SBBD) using the Schrodinger Discover Suite, 1.3) monitor whole cell (MIC) activity, 1.4) track in vitro IC50 potency, 1.5) test ppb, mammalian cytotoxicity, and P-gp inhibition/stimulation, 1.6) FOR, time kill curves, and LpxC inhibitor synergy studies, 1.7) measure PK, 1.8) determine Enterobacteriaceae MIC90, 1.9) synthetic scale up, 1.10) dose-ranging mouse PK/formulation studies, and 1.11) demonstrate in vivo efficacy in a mouse peritonitis model of WT Ec infection. Aim 2 (3 years) - Lead Op to prepare a Pre-Clinical Candidate (PCC) for subsequent safety studies: 2.1) med chem optimization of drug-like properties, 2.2) track MIC, IC50, and cytotoxicity, 2.3) conduct in vitro ADME, 2.4) single mouse (IV, IP, PO) PK, 2.5) validate MOA and FOR, 2.6) test in vitro mammalian off target-activity, 2.7) expanded Enterobacteriaceae MIC90, 2.8) dose-ranging mouse PK, 2.9) synthetic scale up, 2.10) efficacy in murine UTI model, 2.11) identify PK/PD index, 2.12) expanded off target-activity as in 2.6.