Probing the Mechanism of Inactivation of the FOX-4 Cephamycinase by Avibactam

Ceftazidime-avibactam is a second-generation beta-lactam-beta-lactamase inhibitor combination that is effective against Enterobacteriaceae expressing class A extended-spectrum beta-lactamases, class A carbapenemases, and/or class C cephalosporinases. Knowledge of the interactions of avibactam, a diazabicyclooctane with different beta-lactamases, is required to anticipate future resistance threats. FOX family beta-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 blaFOX 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 blaFOX; two isolates, one expressing blaFOX-4 and the other producing blaFOX-5, displayed an MIC of 16 mu g/ml for the combination. FOX-4 possessed a k(2)/K value of 1,800 +/- 100 M-1.s(-1) and an off rate (k(off)) of 0.0013 +/- 0.0003 s(-1). Mass spectrometry showed that the FOX-4-avibactam complex did not undergo chemical modification for 24 h. Analysis of the crystal structure of FOX-4 with avibactam at a 1.5-A resolution revealed a unique characteristic of this AmpC beta-lactamase. Unlike in the Pseudomonas-derived cephalosporinase 1 (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 (dissociation constant) value (1,600 nM) for avibactam compared to other AmpCs (7 to 660 nM). Medicinal chemists must consider the properties of extended-spectrum AmpCs, such as the FOX beta-lactamases, for the design of future diazabicyclooctanes.