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. As current treatments are administered intravenously in a hospital setting, future agents offering oral administration would reduce hospital stay and overall healthcare costs. Entirely new agents with novel mechanisms of action languish; therefore, mechanistically novel antibiotics unaffected by clinically relevant resistance mechanisms and suitable for orally administered stepdown therapy are urgently needed. Our proposal aims to develop a mechanistically novel, IV and PO administered agent to treat infections 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 a small molecule inhibitor series targeting LspA, the essential and broadly conserved Gram-negative lipoprotein signal peptidase that plays a crucial role in outer membrane lipoprotein biogenesis. Preliminary work presented in this proposal establishes the great potential of this series and target. We seek to continue to develop this program through the following aims in a Hit-to-Lead campaign: Aim 1 - Develop toolset to prosecute LspAi Hit-to-Lead campaign. (1) Synthesize 25 mg of LspAi screening hits as well as key intermediates to interrogate nature of the warhead and facilitate analog synthesis in Aim 2. (2) Test LspAis in Ec LspA in vitro biochemical assay to establish in vitro potency. (3) Expand FOR analysis in Ec ΔtolC. (4) Perform FOR and mechanism of action (MOA) analysis in Klebsiella pneumoniae (Kp) ΔtolC to show MOA extends to Kp. Aim 2 – Hit-to-Lead campaign to identify 1 Lead series with WT activity and in vivo efficacy. (1) Hit-to- Lead med chem analog synthesis. (2) MIC determination against WT and matched-pair efflux and permeability modified Ec and Kp. (3) In vitro IC50 determination. (4) Assess plasma protein binding (ppb), mammalian HepG2 cytotoxicity, and hemolytic activity. (5) FOR determination, extended MOA analysis, kill curves and polymyxin synergy assays. (6) Measure PK (IV, SC, and PO). (7) Compound scale-up for in vivo studies. (8) Conduct dose- ranging mouse PK studies. (9) Demonstrate in vivo efficacy in murine septicemia model of WT Ec. Aim 3 - Obtain Ec LspAi-LspA X-ray co-crystal structure in collaboration with SSGCID to enable structure-based drug design approach. SSGCID-led Ec LspA heterologous expression, protein purification, and crystallization studie...