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.

Metallo-β-lactamases (MBLs) are a growing threat to the use of almost all clinically used β-lactam antibiotics. The identification of broad-spectrum MBL inhibitors is hampered by the lack of a suitable screening platform, consisting of appropriate substrates and a set of clinically relevant MBLs. We report procedures for the preparation of a set of clinically relevant metallo-β-lactamases (i.e., NDM-1 (New Delhi MBL), IMP-1 (Imipenemase), SPM-1 (São Paulo MBL), and VIM-2 (Verona integron-encoded MBL)) and the identification of suitable fluorogenic substrates (umbelliferone-derived cephalosporins). The fluorogenic substrates were compared to chromogenic substrates (CENTA, nitrocefin, and imipenem), showing improved sensitivity and kinetic parameters. The efficiency of the fluorogenic substrates was exemplified by inhibitor screening, identifying 4-chloroisoquinolinols as potential pan MBL inhibitors.

Clavulanic acid and avibactam are clinically deployed serine β-lactamase inhibitors, important as a defence against antibacterial resistance. Bicyclic boronates are recently discovered inhibitors of serine and some metallo β-lactamases. Here, we show that avibactam and a bicyclic boronate inhibit L2 (serine β-lactamase) but not L1 (metallo β-lactamase) from the extensively drug resistant human pathogen Stenotrophomonas maltophilia. X-ray crystallography revealed that both inhibitors bind L2 by covalent attachment to the nucleophilic serine. Both inhibitors reverse ceftazidime resistance in S. maltophilia because, unlike clavulanic acid, they do not induce L1 production. Ceftazidime/inhibitor resistant mutants hyperproduce L1, but retain aztreonam/inhibitor susceptibility because aztreonam is not an L1 substrate. Importantly, avibactam, but not the bicyclic boronate is deactivated by L1 at a low rate; the utility of avibactam might be compromised by mutations that increase this deactivation rate. These data rationalize the observed clinical efficacy of ceftazidime/avibactam plus aztreonam as combination therapy for S. maltophilia infections and confirm that aztreonam-like β-lactams plus nonclassical β-lactamase inhibitors, particularly avibactam-like and bicyclic boronate compounds, have potential for treating infections caused by this most intractable of drug resistant pathogens.

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