Inhibitor Affinity Differs among Clinical Variants of IMP Metallo-<b>β</b>-Lactamases: Analysis and Implications for Inhibitor Design

β-Lactam-resistant bacterial infections are a serious concern worldwide. A common mechanism of β-lactam resistance is the expression of β-lactamases, which are capable of hydrolyzing the β-lactam bond in the most commonly used β-lactam antibiotics. Metallo-β-lactamases (MBLs) utilize 1 or 2 zinc ions for catalysis. One of the three most clinically relevant MBLs is Imipenemase (IMP). An important potential way to combat MBLs is to use an inhibitor in combination with an existing β-lactam drug. The current study investigates the mechanism of inhibition of preclinical boronic acid β-lactamase inhibitor RPX 7546 and mercaptomethyl bisthiazolidine D-CS319, which are two previously reported MBL inhibitors, with IMP-1 and its variant IMP-78 (V67F/S262G), chosen due to its improved efficiency hydrolyzing carbapenem β-lactams. A combination of analytical and biochemical experiments and <i>in silico</i> modeling collectively offer a comprehensive understanding of the mechanism of inhibition by these two inhibitors. Our studies show that RPX 7546 is a less effective inhibitor of IMP-78, compared to IMP-1, while D-CS319 shows equally effective inhibition of both enzymes. The findings can be explained in light of the evolution of IMP-78 to overcome structural differences of substrates. Studying inhibitors with variants of clinically relevant MBLs is an area that is growing in importance in the literature. The findings of the current study highlight its significance and the urgent need for the discovery of an MBL inhibitor for clinical use.