Magnolol restores the activity of meropenem against NDM-1-producing Escherichia coli by inhibiting the activity of metallo-beta-lactamase

Abstract

The emergence of plasmid-mediated New Delhi metallo-β-lactamase-1 (NDM-1) in carbapenem-resistant Gram-negative pathogens is an increasing clinical threat. Here we report the discovery of an NDM-1 inhibitor, magnolol, through enzyme inhibition screening. We showed that magnolol significantly inhibited NDM enzyme activity (IC₅₀ = 6.47 µg/mL), and it restored the activity of meropenem against Escherichia coli ZC-YN3, an NDM-1-producing E. coli isolate, in in vitro antibacterial activity assays. Magnolol lacked direct antibacterial activity, but compared with meropenem alone, it reduced the MICs of meropenem against E. coli ZC-YN3 by 4-fold and killed almost all the bacteria within 3 h. Molecular modeling and a mutational analysis demonstrated that magnolol binds directly to the catalytic pocket (residues 110 to 200) of NDM-1, thereby blocking the binding of the substrate to NDM-1 and leading to its inactivation. Our results demonstrate that the combination of magnolol and meropenem may have the potential to treat infections caused by NDM-1-positive, carbapenem-resistant Gram-negative pathogens.

Fig. 1. Magnolol-mediated inhibition of E. coli ZC-YN3 in vitro.

a Chemical structure of magnolol. b Magnolol inhibits NDM-1 activity. The values are the averages of three independent experiments. ** indicates P < 0.01. c Growth curves for E. coli ZC-YN3 cultured with magnolol. Representative data are one of three independent experiments. d Time-kill curves of compounds against E. coli ZC-YN3. The values are the averages of three independent experiments

Fig. 2. The three-dimensional structural determination of the complex formed by NDM-1 with magnolol by the molecular modeling method.

a The structure of the NDM-1-magnolol complex. b The root-mean-square deviations displayed by the backbone atoms of the protein during MD simulations of the NDM-1-magnolol complex (yellow line) and free protein (blue line) are presented. c The number of hydrogen bonds between magnolol and NDM-1 during the simulation

Fig. 3. The predicted interaction mechanism between magnolol and NDM-1.

Decomposition of the binding energy on a per-residue basis in the WT-NDM-1-magnolol complex. The histogram chart shows the total (a), van der Waals (b), electrostatic (c), and solvation (d) contributions for the complexes. The highest occupied molecular orbital (e) and lowest unoccupied molecular orbital (f) of magnolol. g Interactions between magnolol and the residues of the binding sites in NDM-1 are shown using a two-dimensional diagram by Ligplus

Fig. 4. a The effects of WT-NDM-1 and its mutants on NDM-1 activity.

The values are the averages of three independent experiments. * indicates 0.01 < P < 0.05, and ** indicates P < 0.01. b The effects of magnolol on the stability of NDM-1