Exploring a novel Class A β‐Lactamase Inhibitor against the Class C β‐Lactamase Pseudomonas ‐Derived Cephalosporinase (PDC)

Objective To biochemically, structurally, and microbiologically determine the ability and mechanism of 3‐(4‐phenylamino‐phenylamino)‐2‐(1H‐tetrazol‐5‐yl)‐acrylonitrile (FTA) ( Figure 1A ) as an inhibitor of PDC. Hypothesis Given the mechanistic and structural similarities in Class A and Class C β‐lactamases, we hypothesized FTA would bind a modeled hydrophobic allosteric site in PDC analogous to the site it binds in Class A β‐lactamases. Methods Steady state kinetic constants were determined spectrophotometrically using a nitrocefin reporter assay. β‐Lactamase variants were cloned in pET24a+ expression vectors and purified using cation exchange and size exclusion chromatography. Growth curves were determined by measuring OD 600 readings with a microplate reader. Circular dichroism (CD) melting curves and X‐ray crystal structures of both apo and FTA‐bound PDC‐3 WT were determined. Results FTA demonstrates competitive inhibition behavior and does so without time dependence. Apparent inhibition constant ( K I app ) values ( Figure 2B ) for both PDC‐3 WT and PDC‐3 E219K (a variant notable for increased resistance to ceftolozane and ceftazidime, two key anti‐Pseudomonal β‐lactams) were both roughly an order of magnitude lower than in class A β‐lactamases. FTA also impacts the growth kinetics of E. coli expressing PDC‐3 WT in sublethal concentrations of antibiotic ( Figure 2A ), dramatically reducing the slope of growth curves through log phase in the presence of 1000 µM FTA and reducing the lowest concentration of antibiotic preventing pellet formation by five (WT) to six (E219K) dilutions. Importantly, FTA alone does not appear to be toxic to E. coli expressing PDC variants in the absence of an antibiotic (e.g. does not impact growth rates) and CD reveals negligible change in the melting curve of PDC‐3 E219K on the addition of 100 µM FTA and a melting point decrease of less than 1°C for PDC‐3 WT, substantially less than the difference between WT and E219K alone. Consistent with steady state kinetics, X‐ray crystallography ( Figure 1B ) reveals active site density after FTA soaking experiments that could fit an FTA molecule. The nitrile moiety of FTA forms a hydrogen bond with serine 64, the key residue involved in formation of the acyl‐enzyme complex, suggesting a novel inhibition mechanism. Conclusions FTA is a better inhibitor of PDC than class A β‐lactamases and serves as a competitive, active site inhibitor in PDC in contrast to a non‐competitive, allosteric inhibitor in class A. Given that it does not resemble a β‐lactam or a currently available β‐lactamase inhibitor, FTA may serve as a starting point for the development of future β‐lactamase inhibitors. Additionally, these vastly different mechanisms in different classes will serve as the basis of further investigations into the similarities and differences in structure‐activity relationships in class A and class C β‐lactamases.