Structural basis of the UDP-diacylglucosamine pyrophosphohydrolase LpxH inhibition by sulfonyl piperazine antibiotics

Abstract

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.

The Proteobacterial Methanotroph Methylosinus trichosporium OB3b Remodels Membrane Lipids in Response to Phosphate Limitation

“…MED193 ( 18 ), Pseudomonas aeruginosa ( 17 ), and Pelagibacter ubique ( 19 ). LpxH encoding an enzyme involved in lipid A biosynthesis is used as an outgroup ( 49 , 50 ). (E) Neighbor-joining phylogenetic tree showing Agt of strain OB3b, together with characterized GT4-group glycosyltransferases, including Agt from Pelagibacter ubique ( 19 , 23 ), Agrobacterium tumefaciens ( 21 ), and Pseudomonas aeruginosa ( 17 ).…”

Chemical Highlights Supporting the Role of Lipid A in Efficient Biological Adaptation of Gram-Negative Bacteria to External Stresses

“…Additionally, Bohl et al found an IC 50 of 1.2 ± 0.2 μM for 33 against LpxH-mCherry and demonstrated its ability to block the conversion of UDP-DAG to Lipid X . Importantly, it was encountered that 33 was unable to inhibit the LpxH of H. influenzae and P. aeruginosa even at a high concentration up to 100 μM . This may suggest an intrinsic nonsusceptibility due to a difference in the target site or lack of reaching the internal concentration required for action (by permeability barrier or efflux pump).…”

“…171 Importantly, it was encountered that 33 was unable to inhibit the LpxH of H. influenzae and P.aeruginosa even at high concentration up to 100 µM. 169 This may suggest an intrinsic non-susceptibility due to difference in the target site or lack to reach the internal concentration required for action (by permeability barrier or efflux pump).…”

“…Recently, a diffusion disk assay suggested that 33 may be an effective inhibitor of K. pneumoniae LpxH, and Kwak et al report the crystal structure of the K. pneumoniae LpxH/ 33 complex . It was reported that the modification of the phenyl or N-acyl indoline groups of 34 led to compounds able to inhibit the LpxH of K. pneumoniae at a low concentration (0.1 μM).…”

Pharmacoinformatics approaches to identify potential hits against tetraacyldisaccharide 4′-kinase (LpxK) ofPseudomonas aeruginosa

“…Further, through sequence of few biosynthetic steps catalyzed by enzymes such as Kdt A , LpxL and LpxM the lipopolysaccharide KDO 2 -lipid A is formed, where KDO is a 3-deoxy-D-manno-oct-2-ulosonic acid unit. [17][18][19][20][21] Some potent inhibitors of lipopolysaccharide biosynthesis have been reported against LpxA, LpxC, LpxD and LpXH of various bacterial species [22][23][24][25][26][27][28][29][30] (Fig. 2).…”