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

Abstract

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 (SBL). The diazabicyclooctane (DBO) avibactam is a potent AmpC inhibitor; the clinical success of avibactam combined with ceftazidime has stimulated efforts to optimise 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 (AmpCEC) 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 AmpCEC inhibition. The results reveal that, under our assay conditions, zidebactam manifests increased potency (Kiapp 0.69 μM) against AmpCEC compared to the other DBOs (Kiapp 5.0-7.4 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 AmpCEC-zidebactam carbamoyl-complex compared to those for the other DBOs. The results suggest carbamoyl-complex lifetime should be a consideration in development of DBO-based SBL inhibitors for the clinically important class C SBLs.

Studies on enmetazobactam clarify mechanisms of widely used β-lactamase inhibitors

“…However, studies under native-MS conditions imply that formation of this +169-Da species is promoted under high-energy conditions, generated either by collisional activation in the gas phase for MS analysis or by incubation at higher temperature in the aqueous phase, and thus formation of the +169-Da species is likely of limited relevance in physiological conditions. ESI-MS condition influenced fragmentation of the covalent modifications of SBLs by other SBLi has also been reported, e.g., desulfation of avibactam ( 43 , 44 ).…”

New Carbapenemase Inhibitors: Clearing the Way for the β-Lactams

“…The kinetic mechanisms of binding of bicyclic boronates to β-lactamases remain to be established; however, it has been observed that these compounds often display very good activity (IC 50 below the mM range) [74,75]. The results of microbiological assays also support the potential of these compounds as broad spectrum β-lactamase inhibitors [76] (Tables 3-5).…”

Biochemical exploration of β-lactamase inhibitors

“…The reaction of nacubactam produced open ring products, with the piperidine ring assuming a chair conformation ( Figure 6 ) and the carbamoyl-carbonyl oxygen positioned to engage with the backbone NH of A318. Through polar interactions with N346, T316, and K315, the N-sulfate group is 188 securely fixed in the active site ( Lang et al, 2021 ).…”

“…Active regions of AmpC-nacubactam complex where nacubactam was modeled in a single conformation A in which N sulfate is close to Y150. (Adapted from Lang et al, 2021 ).…”

“…Active region of AmpC-relebactam complexes where relebactam was modeled in N-sulfated conformations (A,B) . (Adapted from Lang et al, 2021 ).…”