Novel non-β-lactam inhibitor of β-lactamase TEM-171 based on acylated phenoxyaniline

Abstract

The microbial resistance to antibiotics is a genuine global threat. Consequently, a search of new inhibitors remains of acute importance due to the increasing spread of multidrug resistance. Here we present a new type of non-β-lactam β-lactamase inhibitor PA-34 based on natural phenoxyaniline, identified using computer-assisted screening of scaffolds related to those of known low-affinity inhibitors. The compound displays reversible competitive inhibition of bacterial β-lactamase TEM-171, with a K of 88 μM. Using enzyme kinetics, infra-red spectroscopy, fluorescence quenching and computer docking, we propose that the inhibitor binds at the entrance to the enzyme active site. This is a novel inhibition mechanism compared to binding covalently to the catalytic serine in the active site or non-covalently to the allosteric site. The residues involved in binding the inhibitor are conserved among molecular class A β-lactamases. The identified compound and its proposed binding mode may have a potential for a regulation of the catalytic activity of a wide range of class A β-lactamases. We also hypothesise that the presented route for finding non-β-lactam compounds may be an effective and durable approach for combating bacterial antibiotic resistance.

Mutual influence of secondary and key drug‐resistance mutations on catalytic properties and thermal stability of TEM‐type β‐lactamases

“…All of them represent the analogs of natural forms of β‐lactamases corresponding to various phenotypes. The recombinant proteins were obtained by a method combining periplasmic expression and site‐directed mutagenesis , and they were characterized by approximately the same expression level under similar cultivation conditions.…”

“…The restriction enzyme DpnI was added into the PCR mixture obtained and after incubation at 37 °C for 1 h it was used to transform E. coli DH5α cells. The previously developed pET‐bla expression vector was used as a template. Expression vector pET‐bla encodes the full length β‐lactamase gene including the signal sequence without any tags.…”

The Role of the Ω-Loop in Regulation of the Catalytic Activity of TEM-Type β-Lactamases

“…A new direction involves searching for structural analogues of known βL inhibitors, including allosteric ones. The computational ligand-based in silico screening technique, in combination with molecular docking and experimental studies of βL inhibition, has been applied for the identification of acylated phenoxyaniline and thiourea compounds as novel non-β-lactam inhibitors of TEM-type βLs [11,80]. An analysis of the residues involved in the binding of the inhibitor at the active site of the enzyme showed that some of them are conservative for class A βLs, which can be prospective for inhibitors of these enzymes.…”

“…Computer methods involving the in silico search of novel inhibitors has significantly broadened the range of potential inhibitors. However, only a limited number of novel βL inhibitors have been found that are of non-β-lactam nature and are capable of binding close to the enzyme's active site [8][9][10][11]. Because of the relatively low inhibition constants of such inhibitors, this area of research needs to be further developed.Recently, special attention has been paid to studying the role of loops and peptide linkers as flexible elements in the functioning of proteins and enzymes [12,13].…”

Production of β-Lactamase Inhibitors by Streptomyces Species

“…The discovery of β-lactamase inhibitors introduced new ways to overcome the problem of antibiotic resistance, through drug research and development [ 54 ]. However, as they also contain the same β-lactam ring, they are as susceptible to time-limited application as the β-lactam antibiotics [ 55 ]. Resistance to the β-lactam/β-lactamase inhibitor combinations especially of Gram-negative microorganisms is an old and clinically difficult situation [ 11 , 56 ] that has stimulated the use of computation-based design methods to develop new β-lactamase inhibitors [ 54 ].…”