Dataset Information

Evolution of New Delhi metallo-?-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity.

ABSTRACT: Infections by carbapenem-resistant Enterobacteriaceae are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically used ?-lactamase inhibitors to counter the activity of metallo-?-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-?-lactamase (NDM), which requires a di-nuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact of NDM variants on structure is probed by comparing melting temperature and refolding efficiency and also by spectroscopy (UV-visible, 1H NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining the minimum inhibitory concentrations of various antibiotics, pre-steady-state and steady-state kinetics, inhibitor binding, and zinc dependence of resistance and activity. We observed only minor differences among the fully loaded di-zinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants exhibited improved thermostability (up to ?10 °C increased Tm ) and improved zinc affinity (up to ?10-fold decreased Kd, Zn2). We also provide first evidence that some NDM variants have evolved the ability to function as mono-zinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/Km = 5 × 106 m-1 s-1). These findings reveal the molecular mechanisms that NDM variants have evolved to overcome the combined selective pressures of ?-lactam antibiotics and zinc deprivation.

SUBMITTER: Cheng Z

PROVIDER: S-EPMC6093243 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity.

Cheng Zishuo Z Thomas Pei W PW Ju Lincheng L Bergstrom Alexander A Mason Kelly K Clayton Delaney D Miller Callie C Bethel Christopher R CR VanPelt Jamie J Tierney David L DL Page Richard C RC Bonomo Robert A RA Fast Walter W Crowder Michael W MW

The Journal of biological chemistry 20180616 32

Infections by carbapenem-resistant Enterobacteriaceae are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically used β-lactamase inhibitors to counter the activity of metallo-β-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-β-lactamase (NDM), which requires a di-nuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact ...[more]

PMID: 29909397

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Project description:Bacteria expressing New Delhi metallo-β-lactamase-1 (NDM-1) can hydrolyze β-lactam antibiotics (penicillins, cephalosporins, and carbapenems) and, thus, mediate multidrug resistance. The worldwide dissemination of NDM-1 poses a serious threat to public health, imposing a huge economic burden in the development of new antibiotics. Thus, there is an urgent need for the identification of novel NDM-1 inhibitors from a pool of already-known drug molecules. Here, we screened a library of FDA-approved drugs to identify novel non-β-lactam ring-containing inhibitors of NDM-1 by applying computational as well as in vitro experimental approaches. Different steps of high-throughput virtual screening, molecular docking, molecular dynamics simulation, and enzyme kinetics were performed to identify risedronate and methotrexate as the inhibitors with the most potential. The molecular mechanics/generalized Born surface area (MM/GBSA) and molecular dynamics (MD) simulations showed that both of the compounds (risedronate and methotrexate) formed a stable complex with NDM-1. Furthermore, analyses of the binding pose revealed that risedronate formed two hydrogen bonds and three electrostatic interactions with the catalytic residues of NDM-1. Similarly, methotrexate formed four hydrogen bonds and one electrostatic interaction with NDM-1's active site residues. The docking scores of risedronate and methotrexate for NDM-1 were -10.543 kcal mol-1 and -10.189 kcal mol-1, respectively. Steady-state enzyme kinetics in the presence of risedronate and methotrexate showed a decreased catalytic efficiency (i.e., kcat/Km) of NDM-1 on various antibiotics, owing to poor catalytic proficiency and affinity. The results were further validated by determining the MICs of imipenem and meropenem in the presence of risedronate and methotrexate. The IC50 values of the identified inhibitors were in the micromolar range. The findings of this study should be helpful in further characterizing the potential of risedronate and methotrexate to treat bacterial infections.

Dataset Information

Evolution of New Delhi metallo-?-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity.

Publications

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity.

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