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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 metalation state of NDM within Escherichia 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 metalation state through the imaging of live E. coli using confocal microscopy. NDM-1 is shown to be susceptible to demetalation by intracellular and extracellular metal chelators in a live-cell model of zinc dyshomeostasis, whereas the NDM-15 metalation state is shown to be more resistant to zinc flux. The development of this reversible turn-on fluorescent probe for the metalation state of NDM provides a new tool for monitoring the impact of metal ion sequestration by host defense mechanisms and for detecting inhibitor-target engagement during the development of therapeutics to counter this resistance determinant.

In an effort to probe the biophysical mechanisms of inhibition for ten previously-reported inhibitors of metallo-β-lactamases (MBL) with MBL IMP-1, equilibrium dialysis, metal analyses coupled with atomic absorption spectroscopy (AAS), native state mass spectrometry (native MS), and ultraviolet-visible spectrophotometry (UV-VIS) were used. 6-(1H-tetrazol-5-yl) picolinic acid (1T5PA), ANT431, D/l-captopril, thiorphan, and tiopronin were shown to form IMP-1/Zn(II)/inhibitor ternary complexes, while dipicolinic acid (DPA) and 4-(3-aminophenyl)pyridine-2,6-dicarboxylic acid (3AP-DPA) stripped some metal from the active site of IMP but also formed ternary complexes. DPA and 3AP-DPA stripped less metal from IMP-1 than from VIM-2 but stripped more metal from IMP-1 than from NDM-1. In contrast to a previous report, pterostilbene does not appear to bind to IMP-1 under our conditions. These results, along with previous studies, demonstrate similar mechanisms of inhibition toward different MBLs for different MBL inhibitors.

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 metalation state of NDM within Escherichia 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 metalation state through the imaging of live E. coli using confocal microscopy. NDM-1 is shown to be susceptible to demetalation by intracellular and extracellular metal chelators in a live-cell model of zinc dyshomeostasis, whereas the NDM-15 metalation state is shown to be more resistant to zinc flux. The development of this reversible turn-on fluorescent probe for the metalation state of NDM provides a new tool for monitoring the impact of metal ion sequestration by host defense mechanisms and for detecting inhibitor-target engagement during the development of therapeutics to counter this resistance determinant.