Mechanistic Investigations of Metallo-β-Lactamase Inhibitors: Strong Zinc Binding Is Not Required for Potent Enzyme Inhibition

Metallo-β-lactamases (MBLs) are zinc-dependent bacterial resistance enzymes that can inactivate essentially all classes of β-lactam antibiotics. Infections due to multi-drug-resistant pathogens that express MBLs are difficult to treat and carry high mortality rates. At present there are no clinically approved MBL inhibitors underscoring the urgent need for pharmaceutical agents capable of counteracting the action of these enzymes. In order to develop effective MBL inhibitors it is essential to understand their inhibitory mechanisms. We here describe a comprehensive mechanistic study on a panel of structurally distinct MBL inhibitors drawn from the diverse collection of compounds reported to date in both the scientific and patent literature. Specifically, we determined the half-maximal inhibitory concentration value (IC₅₀) for each inhibitor against purified NDM-1 and IMP-1 revealing clear differences in inhibitory potency among the compounds tested. Additional mechanistic insights into metal binding were also obtained by means of isothermal titration calorimetry (ITC) which was used to assess the affinity of the MBL inhibitors for Zn²⁺, Ca²⁺ and Mg²⁺. These investigations revealed clear differences in metal binding among the MBL inhibitors evaluated. In addition, we directly compared the ability of these compounds to resensitize an NDM-1-expressing E. coli strain to the last resort carbapenem antibiotic meropenem. Notably, indole carboxylate 12 proved to be the most potent inhibitor tested in its ability to synergize with meropenem and with IC₅₀ values in the low nanomolar range against both enzymes. Interestingly, while compound 12 was found the most active MBL inhibitor, it exhibited no appreciable binding to any of the metals tested. These findings provide valuable insights into differences in mechanism and potency for the various classes of MBL inhibitors reported to date.