A kinetic analysis of the inhibition of FOX-4 β-lactamase, a plasmid-mediated AmpC cephalosporinase, by monocyclic β-lactams and carbapenems.
OBJECTIVE
Class C β-lactamases are prevalent among Enterobacteriaceae; however, these enzymes are resistant to inactivation by commercially available β-lactamase inhibitors. In order to find novel scaffolds to inhibit class C β-lactamases, the comparative efficacy of monocyclic β-lactam antibiotics (aztreonam and the siderophore monosulfactam BAL30072), the bridged monobactam β-lactamase inhibitor BAL29880, and carbapenems (imipenem, meropenem, doripenem and ertapenem) were tested in kinetic assays against FOX-4, a plasmid-mediated class C β-lactamase (pmAmpC).


METHODS
The FOX-4 β-lactamase was purified. Steady-state kinetics, electrospray ionization mass spectrometry (ESI-MS) and ultraviolet difference (UVD) spectroscopy were conducted using the β-lactam scaffolds described.


RESULTS
The K(i) values for the monocyclic β-lactams against FOX-4 β-lactamase were 0.04 ± 0.01 μM (aztreonam) and 0.66 ± 0.03 μM (BAL30072), and the Ki value for the bridged monobactam BAL29880 was 8.9 ± 0.5 μM. For carbapenems, the Ki values ranged from 0.27 ± 0.05 μM (ertapenem) to 2.3 ± 0.3 μM (imipenem). ESI-MS demonstrated the formation of stable covalent adducts when the monocyclic β-lactams and carbapenems were reacted with FOX-4 β-lactamase. UVD spectroscopy suggested the appearance of different chromophoric intermediates.


CONCLUSIONS
Monocyclic β-lactam and carbapenem antibiotics are effective mechanism-based inhibitors of FOX-4 β-lactamase, a clinically important pmAmpC, and provide stimulus for the development of new inhibitors to inactivate plasmidic and chromosomal class C β-lactamases.
Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH 44106, USA.
Krisztina M
KM
Papp-Wallace
Susana
S
Mallo
Christopher R
CR
Bethel
Magdalena A
MA
Taracila
Andrea M
AM
Hujer
Ana
A
Fernández
Julian A
JA
Gatta
Kerri M
KM
Smith
Yan
Y
Xu
Malcolm G P
MG
Page
Eric
E
Desarbre
Germán
G
Bou
Robert A
RA
Bonomo
D015780:Carbapenems / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors*; D007700:Kinetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D001618:beta-Lactamases; D047090:beta-Lactams / Q000378:metabolism*
D015780:Carbapenems; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D047090:beta-Lactams; C413413:fox-4 protein, E coli; D001618:beta-Lactamases
bridged monobactams; carbapenems; monosulfactam; β-lactamase inhibitors