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Kinetic and physical studies of beta-lactamase inhibition by a novel penem, BRL 42715.

ABSTRACT: 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.

SUBMITTER: Farmer TH

PROVIDER: S-EPMC1137621 | biostudies-other | 1994 Nov

REPOSITORIES: biostudies-other

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Project description:Antimicrobial resistance is a great public health concern that is now described as a "silent pandemic". The global burden of antimicrobial resistance requires new antibacterial treatments, especially for the most challenging multidrug-resistant bacteria. There are various mechanisms by which bacteria develop antimicrobial resistance including expression of β-lactamase enzymes, overexpression of efflux pumps, reduced cell permeability through downregulation of porins required for β-lactam entry, or modifications in penicillin-binding proteins. Inactivation of the β-lactam antibiotics by β-lactamase enzymes is the most common mechanism of bacterial resistance to these agents. Although several effective small-molecule inhibitors of β-lactamases such as clavulanic acid and avibactam are clinically available, they act only on selected class A, C, and some class D enzymes. Currently, none of the clinically approved inhibitors can effectively inhibit Class B metallo-β-lactamases. Additionally, there is increased resistance to these inhibitors reported in several bacteria. The objective of this study is to use the Resonant Recognition Model (RRM), as a novel strategy to inhibit/modulate specific antimicrobial resistance targets. The RRM is a bio-physical approach that analyzes the distribution of energies of free electrons and posits that there is a significant correlation between the spectra of this energy distribution and related protein biological activity. In this study, we have used the RRM concept to evaluate the structure-function properties of a group of 22 β-lactamase proteins and designed 30-mer peptides with the desired RRM spectral periodicities (frequencies) to function as β-lactamase inhibitors. In contrast to the controls, our results indicate 100% inhibition of the class A β-lactamases from Escherichia coli and Enterobacter cloacae. Taken together, the RRM model can likely be utilized as a promising approach to design β-lactamase inhibitors for any specific class. This may open a new direction to combat antimicrobial resistance.

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Kinetic and physical studies of beta-lactamase inhibition by a novel penem, BRL 42715.

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