Spectroscopic and Biochemical Studies on Metallo‐β‐lactamase IMP‐1

β‐Lactamases are a primarily cause for clinical resistance to β‐lactam antibiotics, such as penicillins, cephamycins, carbapenems, and cephalosporins. Therefore, there are considerable efforts to discover inhibitors of β‐lactamases, which can be given in combination with existing β‐lactams as a treatment for antibiotic resistant bacterial infections. According to previous studies, several types of metallo‐β‐lactamases inhibitors have been discovered by organic chemists: metal‐ion binding inhibitors, covalent inhibitors, allosteric inhibitors, and the inhibitors with uncharacterized mechanisms. The bla IMP gene was initially found in clinical imipenem‐resistant Serratia marcescens and Pseudomonas aeruginosa isolates in Japan. The IMP enzymes deliver broad substrate specificities, and comparing to penicillins they have greater binding affinities for carbapenems and cephalosporins. IMP‐1, the first variant identified, is the most studied IMP variant, and there have been many reported inhibitors of IMP‐1. However, most of these studies reported IC 50 and/or MIC values, and most of the inhibitors have not been characterized for mechanism of inhibition. This lack of understanding of mechanism of inhibition could potentially be reducing our efforts to find effective inhibitors of IMP variants and other metallo‐β‐lactamases. We used spectroscopic and biochemical studies to probe the mechanism of inhibition for several known metallo‐β‐lactamase inhibitors with IMP‐1. Equilibrium dialysis was used to incubate IMP‐1 with different inhibitors, and metal analyses were used to determine the zinc content in the resulting samples. This technique allows for us to determine whether an inhibitor strips zinc from IMP‐1. Native mass spectrometry (Orbitrap) of IMP‐1 samples containing inhibitors were used to confirm the equilibrium dialysis results and also to determine whether an IMP‐1/zinc/inhibitor ternary complex forms. Native mass spectrometry can be used to determine whether inhibitor binds to the zinc‐containing form of IMP‐1, but this technique cannot determine whether the inhibitor(s) bind to the metal ions in the active site. Therefore, we utilize UV‐VIS spectrophotometry with cobalt‐substituted analogs of IMP‐1. This technique can be used to probe whether the inhibitor binds to one or both of the metal ions. We also probe inhibitor binding to the cobalt‐containing enzyme by using Electron Paramagnetic Resonance (EPR) spectroscopy. We have discovered that EPR can be used to determine whether inhibitors bind to both metal ions. By using these techniques, we hope to identify inhibitors that form ternary complexes, rather than those that strip metal ions from the metallo‐β‐lactamases. We believe that the inhibitors that form ternary complexes are the best candidates for rational re‐design efforts. Support or Funding Information National Institutes of Health (NIH) and Miami University