Studies on the mode of action and beta-lactamase inhibitory properties of novel oxapenem compounds

Abstract

Four novel oxapenem compounds were evaluated for their ß-lactamase inhibitory and antibacterial properties. Two (AM-112 and AM-113) displayed intrinsic antibacterial activity with MICs of between 2 to 16µg/ml and 0.5-2µg/ml against Escherichia coli and methicillin-sensitive and -resistant Staphylococcus aureus, respectively. The isomers of these compounds, AM-115 and AM-114 did not display significant antibacterial activity. Combination of the oxapenems with ceftazidime afforded protection against ß-lactamase-producing strains, including hyperproducers of class C enzymes and extended-spectrum ß-lactamase enzymes. A fixed 4µg/ml concentration of AM-112 protected a panel of eight cephalosporins against hydrolysis by class A and class C ß-lactamase producers. In vivo studies confirmed the protective effect of AM-112 for ceftazidime against ß-lactamase producing S. aureus, Enterobacter cloacae and E. coli strains in a murine intraperitoneal infection model. Each of the oxapenems inhibited class A, class C and class D ß-lactamases isolated from whole cells and purified by isoelectric focusing. AM-114 and AM-115 were as effective as clavulanic acid against class A enzymes. AM-112 and AM-113 were less potent against these enzymes. Class C and class D enzymes proved very susceptible to inhibition by the oxapenems. Molecular modelling of the oxapenems in the active site of the class A. TEM-1 and class C P99 enzymes identified a number of potential sites of interaction. The modelling suggested that Ser-130 in TEM-1 and Tyr-150 in P99 were likely candidates for cross-linking of the inhibitor, leading to inhibition of the enzyme. Morphology studies indicated that sub-inhibitory concentrations of the oxapenems caused the formation of round-shaped cells in E. coli DC0, indicating inhibition of penicillin-binding protein 2 (PBP2). The PBP affinity profile of AM-112 was examined in isolated cell membranes of E. coli DC0, S. aureus NCTC 6571, Enterococcus faecalis SFZ and E. faecalis ATCC 29213, in competition with a radiolabelled penicillin. PBP2 was identified as the primary target for AM-112 in E. coli DC0. Studies on S. aureus NCTC 6571 failed to identify a binding target. AM-112 bound to all the PBPs of both E. faecalis strains, and a concentration of 10µg/ml inhibited all the PBPs except PBP3.