Structural Investigations of the Inhibition of Escherichia coli AmpC β-Lactamase by Diazabicyclooctanes

• Published in: Antimicrobial agents and chemotherapy Vol. 65; no. 2
• Main Authors: Lang, Pauline A., Leissing, Thomas M., Page, Malcolm G. P., Schofield, Christopher J., Brem, Jürgen
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 20.01.2021
• Subjects: Anti-Bacterial Agents - pharmacology; Azabicyclo Compounds - pharmacology; Bacterial Proteins - genetics; beta-Lactamase Inhibitors - pharmacology; beta-Lactamases - genetics; Escherichia coli - genetics; Mechanisms of Resistance; Microbial Sensitivity Tests; zidebactam; relebactam; antimicrobial resistance; cephalosporin resistance; avibactam; Avycaz; nacubactam; diazabicyclooctane; serine β-lactamase inhibitors
• ISSN: 0066-4804; 1098-6596; 1098-6596
• DOI: 10.1128/AAC.02073-20

β-Lactam antibiotics are presently the most important treatments for infections by pathogenic Escherichia coli , but their use is increasingly compromised by β-lactamases, including the chromosomally encoded class C AmpC serine-β-lactamases (SBLs). The diazabicyclooctane (DBO) avibactam is a potent AmpC inhibitor; the clinical success of avibactam combined with ceftazidime has stimulated efforts to optimize the DBO core. β-Lactam antibiotics are presently the most important treatments for infections by pathogenic Escherichia coli , but their use is increasingly compromised by β-lactamases, including the chromosomally encoded class C AmpC serine-β-lactamases (SBLs). The diazabicyclooctane (DBO) avibactam is a potent AmpC inhibitor; the clinical success of avibactam combined with ceftazidime has stimulated efforts to optimize the DBO core. We report kinetic and structural studies, including four high-resolution crystal structures, concerning inhibition of the AmpC serine-β-lactamase from E. coli (AmpC EC ) by clinically relevant DBO-based inhibitors: avibactam, relebactam, nacubactam, and zidebactam. Kinetic analyses and mass spectrometry-based assays were used to study their mechanisms of AmpC EC inhibition. The results reveal that, under our assay conditions, zidebactam manifests increased potency (apparent inhibition constant [ K iapp ], 0.69 μM) against AmpC EC compared to that of the other DBOs ( K iapp = 5.0 to 7.4 μM) due to an ∼10-fold accelerated carbamoylation rate. However, zidebactam also has an accelerated off-rate, and with sufficient preincubation time, all the DBOs manifest similar potencies. Crystallographic analyses indicate a greater conformational freedom of the AmpC EC -zidebactam carbamoyl complex compared to those for the other DBOs. The results suggest the carbamoyl complex lifetime should be a consideration in development of DBO-based SBL inhibitors for the clinically important class C SBLs.