Identification of Inhibitors of the Antibiotic-Resistance Target New Delhi Metallo-β-lactamase 1 by both Nanoelectrospray Ionization Mass Spectrometry and Ultrafiltration Liquid Chromatography/Mass Spectrometry Approaches

Abstract

Mass spectrometry-based platforms have gained increasing success in discovery of ligands bound to therapeutic targets as drug candidates. We established both a nanoelectrospray ionization mass spectrometry (nanoESI-MS) assay and an ultrafiltration liquid chromatography/mass spectrometry (LC/MS) assay to identify new ligands for New Delhi metallo-β-lactamase 1 (NDM-1), responsible for worldwide antibiotic resistance. To alleviate nonspecific binding of hydrophobic compounds and eliminate false positives typically encountered in the indirect LC/MS-based assay, we introduced a blocking protein in the control, which remarkably enhances the selectivity and accuracy of the indirect approach. Side-by-side comparison of the two MS-based approaches for the first time further reveals unique advantages of the indirect approach, including better reproducibility and tolerance of interference. Moreover, the success of fishing out a potent ligand from a mixture of small-molecule fragments demonstrates great potential of the indirect LC/MS-based approach for constructing a robust screening platform against combinatorial libraries or natural product extracts. More importantly, by combining the results of MS-based analyses, enzymatic activity assay, competition experiments, and structural simulation, we discovered a new compound as a promising drug candidate targeting NDM-1.

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

“…However, the L-captopril:NDM-1 interaction is well-characterized and can be used to detect any impact that mutations have on ligand binding at the heart of the active site. Isothermal titration calorimetry was used to determine Kd for NDM-1 and L-captopril (Kd = 4 ± 1 µM), and this value is similar to other reports of the affinity of this interaction (Figure 3) (8). In comparison, the dissociation constants for L-captopril with the other NDM variants only occurred within a very narrow range (1.3 to 5 µM), indicating that the mutations derived from the clinic do not greatly impact affinity of an active-site ligand ( Figure S4, Table S5).…”

Mitochondrion‐Targeting Identification of a Fluorescent Apoptosis‐Triggering Molecule by Mass Spectrometry Elucidates Drug Tracking

“…Opening of mPTPs in isolated mitochondria could be detected spectrophotometrically by monitoring the decrease in absorbance at 540 nm ( A 540 ) after overloading mitochondria with high concentrations of Ca 2+ . As expected, NA‐17 could promote mPTP opening (Figure A), even better than high concentrations of Ca 2+ , a typical mPTP‐opening positive control …”

Comparison of analytical techniques for the identification of bioactive compounds from natural products

“…Another approach to prevent excessive binding of compounds to the ultrafiltration membrane was carried out by using a blocking protein. 24 For example, glutathione S -transferase (GST) was demonstrated to reduce non-specific binding and also effectively decreased background signals.…”