β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as ‘transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.

ABSTRACTβ-Lactamase-mediated resistance is a growing threat to the continued use of β-lactam antibiotics. The use of the β-lactam-based serine-β-lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non-β-lactam inhibitor avibactam has extended the utility of β-lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo-β-lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo-β-lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of “dual-action” inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difficult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo-β-lactamases. Here we report that cyclic boronates are able to inhibit all four classes of β-lactamase, including the class A extended spectrum β-lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa, and class D OXA enzymes with carbapenem-hydrolyzing capabilities. We demonstrate that cyclic boronates can potentiate the use of β-lactams against Gram-negative clinical isolates expressing a variety of β-lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other β-lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo-β-lactamase catalysis.

This is a repository copy of Studies on the inhibition of AmpC and other β-lactamases by cyclic boronates.

The class D (OXA) serine β-lactamases are a major cause of resistance to β-lactam antibiotics. The class D enzymes are unique amongst β-lactamases because they have a carbamylated lysine that acts as a general acid/base in catalysis. Previous crystallographic studies led to the proposal that β-lactamase inhibitor avibactam targets OXA enzymes in part by promoting decarbamylation. Similarly, halide ions are proposed to inhibit OXA enzymes via decarbamylation. NMR analyses, in which the carbamylated lysines of OXA-10, -23 and -48 were C-labelled, indicate that reaction with avibactam does not ablate lysine carbamylation in solution. While halide ions did not decarbamylate theC-labelled OXA enzymes in the absence of substrate or inhibitor, avibactam-treated OXA enzymes were susceptible to decarbamylation mediated by halide ions, suggesting halide ions may inhibit OXA enzymes by promoting decarbamylation of acyl-enzyme complex. Crystal structures of the OXA-10 avibactam complex were obtained with bromide, iodide, and sodium ions bound between Trp-154 and Lys-70. Structures were also obtained wherein bromide and iodide ions occupy the position expected for the 'hydrolytic water' molecule. In contrast with some solution studies, Lys-70 was decarbamylated in these structures. These results reveal clear differences between crystallographic and solution studies on the interaction of class D β-lactamases with avibactam and halides, and demonstrate the utility of C-NMR for studying lysine carbamylation in solution.