Molecular and characterization studies of New Delhi metallo-β-lactamase.

Abstract

ABSTRACT The increasing rate of antibiotic resistance within human pathogens has caused global concern. Bacterial strains have employed several strategies to resist β-lactam antibiotics, the most prevalent being the expression of metallo-β-lactases (MBLs). The New Delhi (NDM-1) being the most notorious MBL, is able to hydrolyse most β-lactam antibiotics resulting in multiple-drug resistance. NDM-1 renders the β-lactam antibiotic inactive through cleavage of the amide bond. Zinc ions are a key component for this hydrolysis reaction. There are currently no inhibitors available to combat the detrimental effect of these enzymes. Therefore, identification of potential inhibitors is of primary importance within the scientific community. The aims of this study are to investigate the interactions of a recombinant metallo-β-lactamase protein with metal chelator inhibitors using molecular and quantitative techniques. NDM-1 was cloned into the vector pET302 and over expressed in E. coli BL21 (DE3) cells. Furthermore, this study reports kinetic inhibition and thermodynamics of recombinant NDM-1 against four metal chelators, namely, BP1, BP9, BP10 and BP11. The NDM-1 gene was successfully cloned and expressed in E.coli. The expression yield of the protein was 3.6 mg/L. The kinetic parameters of the recombinant NDM-1 β-lactamase were measured as 29.07 μM and 21.77 s-1 of Km and kcat respectively toward nitrocefin. Inhibition studies showed that the Ki values of BP1, BP9, BP10 and BP11 were 0.24, 0.16, 0.19 and 0.18 μM, respectively. BP9 showed the highest inhibition against NDM-1. Fluorescence studies confirmed that BP9 is a better inhibitor compared to the others, showing a high Ka value (11846.77 μM). The metal chelator BP11, through ITC experiments, demonstrated tight binding for Zn2+. It was also shown in this study that all four compounds inhibit MBLs efficiently. Kinetic studies confirmed that BP9 was the best inhibitor among the other four compounds. Therefore, we can conclude that these potential metal chelators may be used as lead molecules for future drug candidates. Future studies will focus on X-ray crystallography, which involves the study of direct interactions of the drug with specific residues of the protein NDM-1. In addition, a study should be carried out to determine whether the metal chelators have the same effect as in NDM-1 with other MBLs such as IMP, VIM and SPM.