The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is an essential lipid A biosynthetic enzyme that is conserved in the majority of gram-negative bacteria. It has emerged as an attractive novel antibiotic target due to the recent discovery of an LpxH-targeting sulfonyl piperazine compound (referred to as AZ1) by AstraZeneca. However, the molecular details of AZ1 inhibition have remained unresolved, stymieing further development of this class of antibiotics. Here we report the crystal structure of Klebsiella pneumoniae LpxH in complex with AZ1. We show that AZ1 fits snugly into the L-shaped acyl chain-binding chamber of LpxH with its indoline ring situating adjacent to the active site, its sulfonyl group adopting a sharp kink, and its N-CF3–phenyl substituted piperazine group reaching out to the far side of the LpxH acyl chain-binding chamber. Intriguingly, despite the observation of a single AZ1 conformation in the crystal structure, our solution NMR investigation has revealed the presence of a second ligand conformation invisible in the crystalline state. Together, these distinct ligand conformations delineate a cryptic inhibitor envelope that expands the observed footprint of AZ1 in the LpxH-bound crystal structure and enables the design of AZ1 analogs with enhanced potency in enzymatic assays. These designed compounds display striking improvement in antibiotic activity over AZ1 against wild-type K. pneumoniae, and coadministration with outer membrane permeability enhancers profoundly sensitizes Escherichia coli to designed LpxH inhibitors. Remarkably, none of the sulfonyl piperazine compounds occupies the active site of LpxH, foretelling a straightforward path for rapid optimization of this class of antibiotics.

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The UDP‐2,3‐diacylglucosamine pyrophosphatase, LpxH, is an essential enzyme in the biosynthesis of the lipid A, which functions as the hydrophobic anchor of lipopolysaccharides or lipooligosaccharides in the outer leaflet of the outer membrane of Gram‐negative bacteria. This enzyme is conserved in the majority of Gram‐negative bacterial pathogens and is an excellent novel antibiotic target. Here we report the development of a coupled, nonradioactive and colorimetric LpxH assay suitable for high‐throughput analysis of LpxH inhibition and screening. Using this new assay, we have established a pharmacophore model for the recently reported LpxH inhibitor by AstraZeneca (dubbed as AZ1 below). Our crystal structure of Klebsiella pneumoniae LpxH (KpLpxH) in complex with AZ1 shows that AZ1 fits snugly into the L‐shaped acyl chain‐binding chamber of LpxH with its indoline ring situating adjacent to the active site, its sulfonyl group adopting a sharp kink, and its N‐CF3‐phenyl substituted piperazine group reaching out to the far side of the LpxH acyl chain‐binding chamber. Our solution NMR investigation later revealed the presence of a second ligand conformation that was unseen in the crystalline state, delineating a cryptic inhibitor envelope that expands the observed footprint of AZ1 in the LpxH‐bound crystal structure. Utilizing these discoveries, we designed new AZ1‐derivatives that display striking improvement in antibiotic activity over AZ1 against wild‐type K. pneumoniae. Co‐administration with outer membrane permeability enhancers profoundly sensitizes E. coli to our designed LpxH inhibitors. As none of these first sulfonyl piperazine compounds occupied the active site of LpxH, we designed a new series of sulfonyl piperazine analogs and discovered an extended N‐acyl chain analog that additionally occupied the untapped polar binding pocket near the manganese cluster of the LpxH active site. We expect that this work will provide guiding design principles for new LpxH inhibitors and establish important frameworks for the future development of antibiotics against multi‐drug resistant Gram‐negative pathogens