Cyclic Boronates Inhibit All Classes of β-Lactamases

Abstract

β-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.

Keywords: antibiotic resistance; beta-lactamases; beta-lactams; boronate; carbapenemase; inhibitors; metalloenzymes.

FIG 1

(A and B) Outline mechanism of cephalosporin hydrolysis by serine-β-lactamases (A) and metallo-β-lactamases (B). The small-molecule elements of the first tetrahedral intermediate EI¹ is common to both mechanisms. (C) Chemical structures of the two cyclic boronates used in this study. The structure of the proposed common tetrahedral intermediate in the serine and metallo-β-lactamase-catalyzed hydrolysis of β-lactam antibiotics is shown for a cephalosporin substrate (cefalexin). We propose that the cyclic boronates mimic this intermediate.

FIG 2

The conformation of the bicyclic ring core of the cyclic boronates in complex with β-lactamases and PBPs is conserved and mimics the tetrahedral anionic intermediate in cephalosporin hydrolysis. (A) Active-site view from a crystal structure of the CTX-M-15:cyclic boronate 1 complex. Potential hydrogen bonding interactions are represented by dashed lines. (B) Overlay of energy-minimized small-molecule structures of cyclic boronate 2 and a modeled species defined by addition of a hydroxide ion onto the β-lactam carbonyl of cefalexin. Energy minimization was carried out using the MM2 energy minimization function in ChemBio3D Ultra. (C) Overlay of our reported (21) cyclic boronate structures in PDB entries 5FQ9 (cyan, OXA-10:cyclic boronate 1), 5FQB (magenta, BcII:cyclic boronate 2), 5FQC (yellow, VIM-2:2), and 5J8X (orange, PBP-5:2) and our CTX-M-15:1 structure (green). Note that there is variation in the conformations of the side chain, but that of the fused bicyclic ring system is highly conserved in all crystal structures and is likely important for optimal binding of the cyclic boronate inhibitors.