Real‐Time Monitoring of New Delhi Metallo‐β‐Lactamase Activity in Living Bacterial Cells by ¹H NMR Spectroscopy

Abstract

Disconnections between in vitro responses and those observed in whole cells confound many attempts to design drugs in areas of serious medical need. A method based on 1D 1H NMR spectroscopy is reported that affords the ability to monitor the hydrolytic decomposition of the carbapenem antibiotic meropenem inside Escherichia coli cells expressing New Delhi metallo-β-lactamase subclass 1 (NDM-1), an emerging antibiotic-resistance threat. Cell-based NMR studies demonstrated that two known NDM-1 inhibitors, L-captopril and ethylenediaminetetraacetic acid (EDTA), inhibit the hydrolysis of meropenem in vivo. NDM-1 activity in cells was also shown to be inhibited by spermine, a porin inhibitor, although in an in vitro assay, the influence of spermine on the activity of isolated NDM-1 protein is minimal. This new approach may have generic utility for monitoring reactions involving diffusible metabolites in other complex biological matrices and whole-cell settings, including mammalian cells.

Target‐Based Whole‐Cell Screening by 1H NMR Spectroscopy

“…As most compounds in the 96-well plate are either known Class B b-lactamase inhibitors or their derivatives,i ti sn ot surprising that ah igh hit rate of 34 %w as observed. [8] Thestrong and weak inhibitors from our NMRbased screen yielded IC 50 values of 0.51 and 120 mm,similar to those of EDTAa nd l-captopril (IC 50 = 175 mm), respectively. We then determined by NMR spectroscopy the IC 50 values against NDM-1 enzymatic activity in bacterial cells of three screening hits (C10, A5, and B6) that were ranked as weak, medium, and strong, following our previously reported procedure ( Figure 4).…”

“…[8] This finding has led us to design an NMR-based whole cell screening experiment that utilizes 1 HNMR signals from meropenem (the substrate) and hydrolyzed meropenem (the product) as end points to detect enzyme inhibition in the native bacterial cell context ( Figure 1). [8] This finding has led us to design an NMR-based whole cell screening experiment that utilizes 1 HNMR signals from meropenem (the substrate) and hydrolyzed meropenem (the product) as end points to detect enzyme inhibition in the native bacterial cell context ( Figure 1).…”

“…After quenching the reaction, the plate was transferred to an automated NMR screening system to acquire 1D 1 HNMR spectra of each well on the plate. Since EDTAisapotent inhibitor of NDM-1 in E. coli cells (the fifty percent inhibitory concentration, IC 50 ,is 1.6 mm), [8] hardly any product signal is detectable.T hus,these two wells represent positive controls for strong NDM-1i nhibitors. For compounds with signals overlapping with the 0.9 ppm signal (for example,w ell A10 and G10 signals in Figure 3), methyl 1 Hs ignals around 1.1 and 2.9 ppm area can be examined for further validation (Supporting Information, Figure S1).…”

Real‐Time Monitoring of Cancer Cell Metabolism and Effects of an Anticancer Agent using 2D In‐Cell NMR Spectroscopy

“…[7] On the other hand, mammalian metabolites have long been studied "in-cell" using 13 C NMR spectroscopy with perfused cells in a spectrometer.…”

Target‐Based Whole‐Cell Screening by 1H NMR Spectroscopy

“…[8] This finding has led us to design an NMR-based whole cell screening experiment that utilizes 1 H NMR signals from meropenem (the substrate) and hydrolyzed meropenem (the product) as end points to detect enzyme inhibition in the native bacterial cell context (Figure 1). …”