The penem BRL 42715, C6-(N1-methyl-1,2,3-triazolylmethylene)penem, is a potent inhibitor of a broad range of bacterial P-lactamases, including the plasmid-mediated TEM, SHV, OXA, and staphylococcal enzymes, as well as the chromosomally mediated enzymes of Bacteroides, Enterobacter, Citrobacter, Serratia, Morganella, Escherichia, Klebsiella, and Proteus species. The concentration of BRL 42715 needed to reduce the initial rate of hydrolysis of most P-lactamase enzymes by 50% was <0.01 ,ug/ml, which was 10-to 100-fold lower than for other l-lactamase inhibitors. These potent inhibitory activities were reflected in the low concentrations of BRL 42715 needed to potentiate the antibacterial activity of ,-lactamase-susceptible Il-lactams. Concentrations of 0.25 ,ug/ml or less considerably enhanced the activity of amoxicillin against many jI-lactamase-producing strains. The MIC50 (MIC for 50% of strains tested) of amoxicillin for 412 ,3-lactamase-producing members of the family Enterobacteriaceae fell from >128 to 2 ,ug/ml in the presence of 1 jig of BRL 42715 per ml, whereas 5 jig of clavulanic acid per ml brought the MIC5. down to 8 jig/ml. Among these 412 strains were 73 Citrobacter and Enterobacter strains, and 1 ,ig of BRL 42715 per ml reduced the M1C50 of amoxicillin from >128 to 2 ,ug/mI for the 48 cefotaxime-susceptible strains and from >128 to 8 ,ig/ml for the 25 cefotaxime-resistant strains.The ,B-lactamase enzymes form a large and diverse group (3,19) and are recognized as a major cause of bacterial resistance to P-lactam antibiotics (14). This resistance can often be overcome either by using P-lactam antibiotics that are stable to hydrolysis by P-lactamases or by combining labile 3-lactams with enzyme inhibitors which may not themselves be useful antibacterial agents but which have the ability to inactivate P-lactamases. The first ,-lactamase inhibitor to have clinical application was clavulanic acid (8, 16), and formulations of amoxicillin plus clavulanic acid and of ticarcillin plus clavulanic acid are available which protect the antibiotics amoxicillin and ticarcillin from hydrolysis by many P-lactamase-producing organisms. More recently described P-lactamase inhibitors include sulbactam (5) and tazobactam (YTR 830; 1). Clavulanic acid is highly active against a broad range of P-lactamases, including the Ic enzymes produced by Proteus vulgaris and Bacteroides fragilis, but is only weakly active against other class I enzymes. Sulbactam is generally less potent than clavulanic acid (9), although it does have some activity against most class I enzymes, while tazobactam is similar in potency to clavulanic acid and also has moderate activity against the class I enzymes.Studies done in our laboratories on the structural modification of the penem nucleus culminated in the synthesis of compounds containing an alkylidene moiety at the C6 position of the penem ring system (I.

The interactions of Staphylococcus aureus, Bacillus cereus I, TEM, Klebsiella pneumoniae K1 and Enterobacter cloacae P99 beta-lactamases with the novel penem inhibitor BRL 42715 were investigated kinetically and, in some cases, by electrospray mass spectrometry (e.s.m.s.). All the beta-lactamases were rapidly inactivated by BRL 42715, with second-order rate constants ranging from 0.17 to 6.4 microM-1.s-1. The initial stoichiometry of beta-lactamase inhibition was essentially 1:1, with the exception of the K1 enzyme. In this instance about 20 molecules of BRL 42715 were hydrolysed before the enzyme was completely inhibited. Inhibited beta-lactamases did not readily regain activity in the absence of BRL 42715, the half-lives for regeneration of free enzyme ranging from 5 min for the K1 beta-lactamase to over 2 days for the staphylococcal enzyme. Recovery of activity was incomplete for TEM-1, K1 and P99 beta-lactamases, suggesting partitioning of the inhibited enzymes to give a permanently (or at least very stable) inactivated species. Examination of the interactions of the penem with TEM, B. cereus I and P99 beta-lactamases by e.s.m.s. also showed rapid and stoichiometric binding of the inhibitor. In all cases a mass increase of 264 Da over the native enzyme was observed, corresponding to the molecular mass of BRL 42715, showing that no fragmentation of the penem occurred on reaction with the beta-lactamases.

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