Probing the Mechanism of Inactivation of the FOX-4 Cephamycinase by Avibactam [PublishAheadOfPrint]

Ceftazidime-avibactam is a "second generation" β-lactam-β-lactamase inhibitor combination that is effective against Enterobacteriaceae expressing class A extended-spectrum β-lactamases, class A carbapenemases and/or class C cephalosporinases. Knowledge of the interactions of avibactam, a diazabicyclooctane with different β-lactamases is required to anticipate future resistance threats. FOX family β-lactamases possess unique hydrolytic properties with a broadened substrate profile to include cephamycins, partly as a result of an isoleucine at position 346, instead of the conserved asparagine found in most AmpCs. Interestingly, a single amino acid substitution at N346 in the Citrobacter AmpC is implicated in resistance to the aztreonam-avibactam combination. In order to understand how diverse active site topologies affect avibactam inhibition, we tested a panel of clinical Enterobacteriaceae isolates producing bla_FOX using ceftazidime-avibactam, determined the biochemical parameters for inhibition using the FOX-4 variant, and probed the atomic structure of avibactam with FOX-4. Avibactam restored susceptibility to ceftazidime for most isolates producing bla_FOX; two isolates expressing bla_FOX-4 or bla_FOX-5 displayed an MIC of 16 μg/mL for the combination. FOX-4 possessed a k₂/K value of 1,800 ± 100 M^-1s^-1 and a k_off of 0.0013 ± 0.0003 s^-1. Mass spectrometry showed that the FOX-4-avibactam complex was stable for 24 hr. Analysis of the crystal structure of FOX-4 with avibactam at a 1.5 Å resolution revealed a unique characteristic of this AmpC β-lactamase. Unlike in the PDC-1 avibactam crystal structure, interactions (e.g., hydrogen bonding) between avibactam and position I346 in FOX-4 are not evident. Furthermore, another residue is not observed to be close enough to compensate for the loss of these critical hydrogen bonding interactions. This observation supports findings from the inhibition analysis of FOX-4; FOX-4 possessed the highest K_d value (1,600 nM) for avibactam compared to other AmpCs (7-660 nM). Medicinal chemists must consider the properties of extended-spectrum AmpCs, such as the FOX β-lactamases for the design of future diazabicyclooctanes.

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