A kinetic analysis of the inhibition of FOX-4 β-lactamase, a plasmid-mediated AmpC cephalosporinase, by monocyclic β-lactams and carbapenems.

Objectives: Class C beta-lactamases are prevalent among Enterobacteriaceae; however, these enzymes are resistant to inactivation by commercially available beta-lactamase inhibitors. In order to find novel scaffolds to inhibit class C beta-lactamases, the comparative efficacy of monocyclic beta-lactam antibiotics (aztreonam and the siderophore monosulfactam BAL30072), the bridged monobactam beta-lactamase inhibitor BAL29880, and carbapenems (imipenem, meropenem, doripenem and ertapenem) were tested in kinetic assays against FOX-4, a plasmid-mediated class C beta-lactamase (pmAmpC). Methods: The FOX-4 beta-lactamase was purified. Steady-state kinetics, electrospray ionization mass spectrometry (ESI-MS) and ultraviolet difference (UVD) spectroscopy were conducted using the beta-lactam scaffolds described. Results: The K-i values for the monocyclic beta-lactams against FOX-4 beta-lactamase were 0.04 +/- 0.01 mu M(aztreonam) and 0.66 +/- 0.03 mu M (BAL30072), and the K-i value for the bridged monobactam BAL29880 was 8.9 +/- 0.5 mu M. For carbapenems, the K-i values ranged from 0.27 +/- 0.05 mu M (ertapenem) to 2.3 +/- 0.3 mu M (imipenem). ESI-MS demonstrated the formation of stable covalent adducts when the monocyclic beta-lactams and carbapenems were reacted with FOX-4 beta-lactamase. UVD spectroscopy suggested the appearance of different chromophoric intermediates. Conclusions: Monocyclic beta-lactam and carbapenem antibiotics are effective mechanism-based inhibitors of FOX-4 beta-lactamase, a clinically important pmAmpC, and provide stimulus for the development of new inhibitors to inactivate plasmidic and chromosomal class C beta-lactamases.