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

beta-Lactam antibiotics are presently the most important treatments for infections by pathogenic Escherichia coli, but their use is increasingly compromised by beta-lactamases, including the chromosomally encoded class C AmpC serine-beta-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-beta- lactamase from E. coli (AmpC(EC)) by clinically relevant DBO-based inhibitors: avibactam, relebactam, nacubactam, and zidebactam. Kinetic analyses and mass spectrometrybased assays were used to study their mechanisms of AmpCEC inhibition. The results reveal that, under our assay conditions, zidebactam manifests increased potency (apparent inhibition constant [K-iapp], 0.69 mu M) against AmpCEC compared to that of the other DBOs (K-iapp 5 5.0 to 7.4 mu M) 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.