The efficacy of β-lactam antibiotics is threatened by the emergence and global spread of metallo-β-lactamase-(MBL) mediated resistance, specifically New Delhi-Metallo-β-lactamase-1 (NDM-1). Utilizing fragment-based drug discovery (FBDD), a new class of inhibitors for NDM-1 and two related β-lactamases, IMP-1 and VIM-2, was identified. Based on 2,6-dipicolinic acid (DPA), several libraries were synthesized for structure-activity relationship (SAR) analysis. Inhibitor 36 (IC50 = 80 nM) was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes. While DPA displayed a propensity to chelate metal ions from NDM-1, 36 formed a stable NDM-1:Zn(II):inhibitor ternary complex, as demonstrated by 1H NMR, electron paramagnetic resonance (EPR) spectroscopy, equilibrium dialysis, intrinsic tryptophan fluorescence emission, and UV-Vis spectroscopy. When co-administered with 36 (at concentrations non-toxic to mammalian cells), the minimum inhibitory concentration (MIC) of imipenem against clinical isolates of Eschericia coli and Klebsiella pneumoniae harboring NDM-1 were reduced to susceptible levels.

Use and misuse of antibiotics has driven the evolution of serine β-lactamases to better recognize new generations of β-lactam drugs, but the selective pressures driving evolution of metallo-β-lactamases are less clear. Here, we present evidence that New Delhi Metallo-β-lactamase (NDM) is evolving to overcome the selective pressure of zinc(II) scarcity. Studies of NDM-1, NDM-4 (M154L), and NDM-12 (M154L, G222D) demonstrate that the point mutant M154L, contained in 50% of clinical NDM variants, selectively enhances resistance to the penam ampicillin at low zinc(II) concentrations relevant to infection sites. Each of the clinical variants is shown to be progressively more thermostable and to bind zinc(II) more tightly than NDM-1, but a selective enhancement of penam turnover at low zinc(II) concentrations indicates that most of the improvement derives from catalysis rather than stability. X-ray crystallography of NDM-4 and NDM-12, as well as bioinorganic spectroscopy of dizinc(II), zinc(II)/cobalt(II), and dicobalt (II) metalloforms probe the mechanism of enhanced resistance and reveal perturbations of the dinuclear metal cluster that underlie improved catalysis. These studies support the proposal that zinc(II) scarcity, rather than changes in antibiotic structure, is driving the evolution of new NDM variants in clinical settings.

New Delhi Metallo-β-lactamase (NDM) grants resistance to a broad spectrum of β-lactam antibiotics including last-resort carbapenems and is emerging as a global antibiotic resistance threat. Limited zinc availability adversely impacts the ability of NDM-1 to provide resistance, but a number of clinical variants have emerged that are more resistant to zinc scarcity (e.g., NDM-15). To provide a novel tool to better study metal ion sequestration in host-pathogen interactions, we describe the development of a fluorescent probe that reports on the dynamic metallation state of NDM within E. coli. The thiol-containing probe selectively coordinates the dizinc metal cluster of NDM and results in a 17-fold increase in fluorescence intensity. Reversible binding enables competition and time-dependent studies that reveal fluorescence changes used to detect enzyme localization, substrate and inhibitor engagement, and changes to metallation state through the imaging of live E. coli using confocal microscopy. NDM-1 is shown to be susceptible to demetallation by intracellular and extracellular metal chelators in a live-cell model of zinc dyshomeostasis, whereas the NDM-15 metallation state is shown to be more resistant to zinc flux. The development of this reversible turn-on fluorescent probe for the metallation state of NDM provides a new tool for monitoring the impact of metal ion sequestration by host defense mechanisms and to detect inhibitor target engagement during the development of therapeutics to counter this resistance determinant.

Metallo‐Beta‐Lactamases (MBLs) confer resistance to carbapenems, cephalosporins, and penicillins in several clinically relevant Gram‐negative bacteria including Acinetobacter, Pseudomonas aeruginosa, and many Enterobacteriaceae. New Delhi Metallo‐Beta‐Lactamases (NDMs) are amongst some of the most worrisome and prevalent MBLs. This family of MBLs is capable of hydrolyzing all generations of bicyclic beta‐lactams. Two new NDM variants, NDM‐4 and ‐12, have recently been identified in E. coli clinical isolates. NDM‐4 contains an M154L substitution while NDM‐12 bears both M154L and G222D mutations. Considering the clinical significance of Gram‐negative species possessing these enzymes, there is a need to characterize the structures and activities of these variants in order to provide a better basis for drug discovery efforts. We have found that NDM‐4 and NDM‐12 possess higher hydrolytic activity compared to NDM‐1. Our differential scanning fluorimetry studies suggest that the M154L mutation confers increased thermal stability and improved affinity for active site catalytic Zn metal ions. The additional G222D mutation of NDM‐12 further improves stability and Zn affinity. M154L, found in the secondary coordination sphere, accounts for changes in stability by acting as a buttress to the loop containing His122, a direct Zn coordinating residue. G222D, on the other hand, is a mutation located in a loop just above the active site approximately 10 Å from the active site zinc ions. Together, our crystal structures of these NDM variants, including the first known structure of NDM‐12, biochemical analyses, and biophysical analyses provide a basis for understanding the roles of key residues surrounding the active site within NDM variants. A better understanding of clinically relevant NDM variants is essential in the fight against MBL‐mediated antibiotic resistance. Our structural, biochemical, and biophysical studies will help to inform drug design efforts to develop new MBL inhibitors.Support or Funding InformationThe authors acknowledge financial support from the US National Institutes of Health (Award No. R01 GM111926 to RCP, DLT, MWC, and WF) and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.

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