Spectroscopic and biochemical characterization of metallo-β-lactamase IMP-1 with dicarboxylic, sulfonyl, and thiol inhibitors

The three decades of investigating β-lactamases inhibitors have resulted in many MBL inhibitors in scientific community; however, the most of the published inhibitors are limited by IC50/MIC values. In this study, equilibrium dialysis, metal analyses coupled with atomic absorption spectroscopy (AAS), native state mass spectroscopy (native MS), and ultraviolet-visible spectroscopy (UV-VIS) were used to characterize the mechanism of inhibition of 10 previously-reported MBL inhibitors with metallo-β-lactamase IMP-1. AC10-4, ANT431, captopril, thiorphan, and tiopronin were shown to form IMP-1/Zn(II)/inhibitor tertiary complexes. DPA and compound 36 showed binding between Zn-IMP-1 and ZnZn-IMP-1 from native state mass spectroscopy. EDTA and 2,3-dimercaprol appear to strip Zn(II) from the active site of IMP-1, and psterostilbene showed no binding with IMP-1 from our study. [Display omitted] •1T5PA, ANT431, captopril, thiorphan, and tiopronin form ternary complexes with metallo-β-lactamase IMP-1.•DPA and 3PA-DPA remove zinc ions from IMP-1 but not as strong as EDTA.•Different metallo-β-lactamase inhibitors do not interact identically with all metallo-β-lactamases. 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.