Kinetic study of interaction between BRL 42715, beta-lactamases, and D-alanyl-D-alanine peptidases

Abstract

A detailed kinetic study of the interactions between BRL 42715, a ␤-lactamase-inhibiting penem, and various ␤-lactamases (EC 3.5.2.6) and D-alanyl-D-alanine peptidases (DD-peptidases, EC 3.4.16.4) is presented. The compound was a very efficient inactivator of all active-site serine ␤-lactamases but was hydrolyzed by the class B, Zn 2؉-containing enzymes, with very different k cat values. Inactivation of the Streptomyces sp. strain R61 extracellular DD-peptidase was not observed, and the Actinomadura sp. strain R39 DD-peptidase exhibited a low level of sensitivity to the compound.The production of ␤-lactamases by clinical isolates continues to be the main mechanism of bacterial resistance to ␤-lactam antibiotics. Whilst antibacterial therapy with ␤-lactamase-stable compounds has been partially successful in containing the problem, absolute stability has not been achieved with any one agent. Clinical experience has established that a second approach, using a specific ␤-lactamase inhibitor coadministered with a ␤-lactamase-labile antibiotic, offers a viable therapeutic option for combatting ␤-lactamase-mediated resistance. Although the three ␤-lactamase inhibitors in clinical use (clavulanic acid, sulbactam, and tazobactam) are effective inactivators of a wide range of ␤-lactamases, they do not inhibit all of the clinically important enzymes (4, 25).Of recent interest is the penem ␤-lactamase inhibitor, BRL 42715 (Fig. 1A), which is synthesized following a stereocontrolled reaction sequence starting from the chiral synthon, 6-aminopenicillanic acid (23). BRL 42715 alone possesses no appreciable antibacterial activity (MIC, 16 to 64 g/ml) except against the methicillin-sensitive staphylococci (MIC, 1 g/ml). In cell-free ␤-lactamase assays, this inhibitor shows intrinsic activity which is superior to that of clavulanic acid, sulbactam, or tazobactam. Significantly, it is able to inactivate the plasmidmediated OXA-1 ␤-lactamase and the chromosomally mediated enzymes of Enterobacter, Citrobacter, Pseudomonas, Serratia, and Morganella species, against which the other inhibitors show poor activity. Its potent inhibitory activity is reflected in its ability to potentiate the antibacterial properties of a wide range of ␤-lactamase-susceptible ␤-lactam antibiotics, including third generation cephalosporins, at very low concentrations. Higher concentrations (Ϸ1 g/ml) are required, however, against those strains producing elevated levels of class C ␤-lactamases (5, 7). The BRL 42715 diffusion rate through the outer membranes of gram-negative bacteria is similar to those of sulbactam and tazobactam but lower than that of clavulanic acid (11). However, thanks to its superior intrinsic activity, it remains the most active inhibitor against whole cells. In contrast to other penems, it is not readily hydrolyzed by the renal dehydropeptidase (6).The kinetics of inhibition of ␤-lactamases of Staphylococcus aureus NCTC 11561 (class A), Escherichia coli JT4 (TEM-1, class A), and Enterobacter cloacae P99 (class C) by BRL 42715 has been...

Three Decades of β-Lactamase Inhibitors

“…IC 50 s of BRL 42715 were 10-to 100-fold lower than those of clavulanate, tazobactam, and sulbactam for class A TEM-1 and SHV-1, class C P99, class D OXA-1, and the S. aureus ␤-lactamase NCTC 11561 (87). However, BRL 42715 was a substrate for class B metallo-␤-lactamases from Aeromonas hydrophila and S. maltophilia and for BcII (250). In susceptibility testing, BRL 42715 concentrations of 0.25 g/ml or lower were able to restore amoxicillin MICs to Ͻ16 g/ml for TEM-1-and OXA-1-producing organisms (87).…”

“…Accordingly, mass spectrometry studies demonstrated that the methylidene penem inhibition pathway does not involve fragmentation in the active site (119,250,406). When TEM-1, P99, and the class A ␤-lactamase from B. cereus I were inactivated by BRL 42715, spectra showed a mass increases equivalent to the molecular masses of the inhibitor plus the enzyme (119).…”

“…Most ␤-lactamases appear to follow this linear pathway to inactivation by the penem compounds, i.e., formation of an acyl-enzyme and rearrangement to the stable dihydrothiazepine ring adduct (119,250). However, some class A enzymes, such as the ␤-lactamases from Staphylococcus albus (Staphylococcus epidermidis) and K. pneumoniae K1, exhibit more complex kinetics (46,250).…”

“…Initial studies to elucidate the BRL 42715 inactivation product using UV difference spectroscopy for both enzyme-and basecatalyzed hydrolysis of the methylidene penem suggested the presence of a dihydrothiazepine rearrangement product (46,250). Accordingly, mass spectrometry studies demonstrated that the methylidene penem inhibition pathway does not involve fragmentation in the active site (119,250,406).…”

“…However, some class A enzymes, such as the ␤-lactamases from Staphylococcus albus (Staphylococcus epidermidis) and K. pneumoniae K1, exhibit more complex kinetics (46,250). A branched pathway leads to an acyl-enzyme hydrolysis product, which also rapidly rearranges to the dihydrothiazepine species.…”