Abstract In 2013, the CDC designated carbapenem-resistant Enterobacteriaceae (CRE) an Urgent Threat, and in 2017, the WHO designated it a Priority 1 “critical superbug”. As few therapies remain to treat CRE, the risk of “pan- resistant” CRE, untreatable by any currently available antibiotic, increases. Entirely new agents with novel mechanisms of action (MOA) not cross-resistant to SOC agents languish. Our proposal aims to develop an O- antigen (O-a) biosynthetic inhibitory agent that potentiates serum-mediated killing (SMK) and is efficacious in a CRE rodent model of infection. We have shown that the O-a biosynthesis gene wecA, a nonessential gene under standard growth conditions, is essential for growth and pathogenesis in the presence of mammalian serum. Our proposal outlines a plan to develop synthetic inhibitors of WecA that we previously discovered and optimized to inhibit the Gram-positive WecA ortholog, TarO. Our Aims are: Aim 1 (Phase 1; Ph1). Screening, MOA studies, and Proof-of-Concept in vivo Studies. (1) Complete lux reporter screening of the tarocin focused library for SMK against WT and ΔtolC E. coli (Ec), (2) directly confirm tarocins inhibit EcWecA as their MOA for eliciting SMK, (3) demonstrate that tarocin-induced SMK extends to Kp, and (4) demonstrate proof-of-concept in vivo efficacy. Milestone 1. Screen ~600 additional tarocin analogs for SMK activity and identify up to 4 chemically distinct tarocin subseries demonstrating i) > 4-fold EC50 shift in SMK by EcWecA overexpression, ii) dose-dependent depletion of O-a in a whole-cell context, iii) causal drugR mutations mapping to EcwecA, iv) SMK activity against Kp DtolC, v) >90% HepG2 cell viability at 25X MIC, and vi) favorable 50% protective dose for survival in a rat septicemia model using an efflux-deficient Ec strain. Aim 2 (Ph2). Lead ID/Opt. Identify tarocins with potent WT Ec and Kp SMK by (1) empirically testing analogs for SMK against panel of Ec/Kp permeability/efflux deficient mutants, (2) employing recent physicochemical rules of GN entry, and (3) exploring siderophore conjugation to drive SAR efforts. We will also optimize PK and drug- like properties. Milestone 2. Identify up to 3 analogs demonstrating i) Ec/Kp WT activity (MIC in serum [MICs] <1 ug/ml), ii) PK exposure to cover MICs for 4 hrs, iii) Ec/Kp target/pathway engagement selectivity including tarocinR wecA mutation in Kp, v) Ec/Kp FOR <1x10-9 at 8X MICs, vi) MICs90 <2 ug/ml (100 isolates), and vii) > 90% HepG2/HEK293 viability at 50X MICs, clean vs CYP/ion channels (>10 uM), and PanLabs IC50 >10 uM. Aim 3 (Ph2). In vivo efficacy demonstration. (1) Optimize synthetic routes to efficiently prepare 3 analogs for formulation and dose-ranging rat PK studies, (2) identify formulation vehicles for the 3 analogs to enable oral and IV PK dosing in in vivo studies, and (3) demonstrate in vivo efficacy for our top compound in a rodent septicemia model using Ec and Kp strains. Milestone 3. Synthesize 500 mg (>95% p...