The rapid emergence of antibiotic-resistant bacteria is a major global health threat. This spurs the need to revisit key antibacterial drug targets such as the peptidoglycan (PG) layer. The PG synthesis machinery is targeted by β-lactam antibiotics that inhibit penicillin-binding proteins (PBP) which crosslink PG strands. A main resistance mechanism is the expression of β-lactamases that can degrade β-lactams. There is thus a critical need for new antibiotics or for avenues to re-sensitize bacteria to β-lactam antibiotics. For the latter, one approach is developing β-lactamase inhibitors; unfortunately, the 5 current inhibitors do not inhibit certain key β-lactamases, and there are resistance mechanisms via inhibitor-resistant β-lactamases. A second approach is to inhibit PG degrading lytic transglycosylases (LT), the focus of this application. Inhibition of LTs or knocking out LTs genetically has been shown to restore the efficacy of β-lactam antibiotics in many serious pathogens including Escherichia coli, Neisseria meningitides, Pseudomonas aeruginosa, Enterobacter aerogenes, Acinetobacter baumannii, Helicobacter pylori, and Campylobacter jejuni. This β-lactam potentiation involves two possible mechanisms of which, depending on the pathogen, either or both contribute. In the first mechanism, the inhibition of both PBP and LT leads to long non-cross-linked PG strands that cause cell wall bulges, weakening the cell wall. In the second mechanism, LT activity generates disaccharide PG product that, when recycled to the cytoplasm, increases β-lactamase expression in certain pathogens. Despite these compelling observations, there is only one promising LT inhibitor known, bulgecin A; however, this natural product carbohydrate-based inhibitor is very challenging for medicinal chemistry efforts. As a result, bulgecin A has not been very amenable to advancing inhibition studies towards animal studies and beyond. This application proposes to overcome this key roadblock by developing new LT inhibitors with scaffold(s) different from bulgecin A via biased (Aim 1) and non-biased fragment-based approaches (Aim 2). Aim 1: To identify new inhibitor fragments that retain bulgecin A's key N-acetyl group. N-acetyl containing compounds will be selected or designed aided by docking; their LT binding and inhibition will be probed by biophysical techniques, protein crystallography, and enzymatic assays. Compounds will be tested against multiple LTs known to bind bulgecin A, and which are amenable to crystallography (E. coli, P. aeruginosa, and C. jejuni) in order to identify at least one fragment binding to one LT as a novel starting point for optimization. Aim 2: To identify non-acetyl containing fragments that bind to the active site of LT, we will screen non- biased fragments against LTs for binding and inhibition as in Aim 1. Such fragments could bind to the N-acetyl binding pocket or to the adjacent pockets. Compounds will be obtained from an sp3 fragment lib...