Abstract
The New Delhi metallo-β-lactamase (NDM-1) is an important clinical target for antimicrobial research, but there are insufficient clinically useful inhibitors and the details of NDM-1 enzyme catalysis remain unclear. The aim of this work is to provide a thermodynamic profile of NDM-1 catalysed hydrolysis of β-lactams using an isothermal titration calorimetry (ITC) approach and to apply this new method to the identification of new low-molecular-weight dicarboxylic acid inhibitors. The results reveal that hydrolysis of penicillin G and imipenem by NDM-1 share the same thermodynamic features with a significant intrinsic enthalpy change and the release of one proton into solution, while NDM-1 hydrolysis of cefazolin exhibits a different mechanism with a smaller enthalpy change and the release of two protons. The inhibitory constants of four carboxylic acids are found to be in the micromolar range. The compounds pyridine-2,6-dicarboxylic acid and thiazolidine-2,4-dicarboxylic acid show the best inhibitory potency and are confirmed to inhibit NDM-1 using a clinical strain of Escherichia coli The pyridine compound is further shown to restore the susceptibility of this E. coli strain to imipenem, at an inhibitor concentration of 400 μM, while the thiazoline compound also shows a synergistic effect with imipenem. These results provide valuable information to enrich current understanding on the catalytic mechanism of NDM-1 and to aid the future optimisation of β-lactamase inhibitors based on these scaffolds to tackle the problem of antibiotic resistance.