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.
Cutaneous lupus erythematous (CLE) is a disfiguring manifestation of systemic LE (SLE). The pathophysiology of CLE is unclear. However, regulation of skin inflammation and apoptosis contribute, potentially in a female-biased manner. Differential DNA methylation is important in the organ-specific manifestations of SLE but has not been studied in skin. Thus, we explored the genome-wide DNA methylation changes in keratinocytes (KC) to investigate the functional relevance in CLE. Analysis of cultured KC (at passage 2) from 8 patients with SLE and 8 age, sex, and ethnicity matched controls was performed using the Infinium MethylationEPIC array and investigated biological significance using DAVID database. We identified 1443 differentially methylated sites with 924 hypomethylated genes and 519 hypermethylated genes in lupus KC compared to controls. The top canonical pathway was Hippo signaling, which is key in promoting apoptosis. TEA Domain Transcription Factor 1 (TEAD1), a transcription factor in the Hippo pathway, was significantly hypomethylated in lupus compared to control (Db [-0.17, P¼ 4.36 X 10-9), which could promote cell death. Further, methylation of LATS1/2, which inactivates TAZ and YAP, members of the Hippo signaling pathway, was significantly increased (Db¼ 0.11, P¼ 3.82 X 10-4 possibly leading to increased YAP/TAZ inhibition and increased apoptosis. YAP and TAZ were both hypermethylated (Db¼ 0.11 (P¼ 1.53 X 10-3) and 0.12 (P¼ 2.20 X 10-4)), thereby increasing TEAD1 activity. Vestigial like family member 3 (VGLL3) has been identified as a putative transcription factor and orchestrator of sex bias in autoimmune diseases. Importantly, VGLL3 operates through TEAD1 in other organs, and may be an upstream regulator in the skin. Overall, these results suggest that differential methylation in KC may underly dysregulated apoptosis and female bias of CLE.
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