Interaction of Avibactam with Class B Metallo-β-Lactamases

Abstract

β-Lactamases are the most important mechanisms of resistance to the β-lactam antibacterials. There are two mechanistic classes of β-lactamases: the serine β-lactamases (SBLs) and the zinc-dependent metallo-β-lactamases (MBLs). Avibactam, the first clinically useful non-β-lactam β-lactamase inhibitor, is a broad-spectrum SBL inhibitor, which is used in combination with a cephalosporin antibiotic (ceftazidime). There are multiple reports on the interaction of avibactam with SBLs but few such studies with MBLs. We report biochemical and biophysical studies on the binding and reactivity of avibactam with representatives from all 3 MBL subfamilies (B1, B2, and B3). Avibactam has only limited or no activity versus MBL-mediated resistance in pathogens. Avibactam does not inhibit MBLs and binds only weakly to most of the MBLs tested; in some cases, avibactam undergoes slow hydrolysis of one of its urea N-CO bonds followed by loss of CO2, in a process different from that observed with the SBLs studied. The results suggest that while the evolution of MBLs that more efficiently catalyze avibactam hydrolysis should be anticipated, pursuing the development of dual-action SBL and MBL inhibitors based on the diazabicyclooctane core of avibactam may be productive.

FIG 1

Outlines of reaction schemes for β-lactamase hydrolysis and the reactions of avibactam with SBLs and MBLs. (A) Reactions catalyzed by serine β-lactamases (SBLs) and metallo-β-lactamases (MBLs), outlining proposed intermediates. Enz, enzyme. (B) Note the variation in mechanisms for avibactam hydrolysis as catalyzed by SBLs and MBLs. With SBLs (with KPC-2), the available evidence is that hydrolysis of the acyl-enzyme complex proceeds via initial elimination to give an imine followed by acyl-enzyme ester hydrolysis (11). In the case of the MBLs, ester hydrolysis can occur without elimination and imine hydrolysis.

FIG 2

Studies of the binding of avibactam to MBLs using NMR. (A) ¹H CPMG analyses of avibactam binding to selected MBLs imply that avibactam is a weak binder of the indicated MBLs as evidenced by a reduction of the signal intensity of avibactam when in the presence of an added MBL in solution. (B) ¹⁹F-NMR analysis of avibactam of NDM-1* indicates that avibactam either binds NDM-1* near its active loop or its binding induces changes in the labeled L1 loop (28). For assay conditions, see Materials and Methods.

FIG 3

Analyses of the hydrolysis reactions of avibactam with selected MBLs by NMR and Raman spectroscopy. (A) 2D ¹H-¹³C correlation analysis of a mixture of 50% avibactam- and 50% avibactam-hydrolyzed products. Edited HSQC: CH (red), CH₂ (pink). HMBC: avibactam pure (green), hydrolysis-avibactam reaction mixture 50:50 (blue). Hydrolysis product signals are in the black boxes in the 2D ¹H-¹³C correlation. The 1D ¹H spectrum displayed at the top of the 2D plane corresponds to avibactam (red) and the hydrolysis-avibactam reaction mixture 50/50 (blue). The black labels correspond to the hydrolyzed products. Orange labels correspond to avibactam. (B) Assigned chemical shifts (parts per million) of the products of avibactam VIM-4-mediated hydrolysis are consistent with the products observed with SPM-1 and NDM-1 (2D characterization data not shown). (C) Raman spectroscopic analyses reveal that VIM-4-catalyzed hydrolysis of avibactam proceeds predominantly via simple hydrolysis (and loss of CO₂) of the avibactam cyclic urea (Fig. 1). Blue trace, VIM-4 and avibactam after 240 min; red trace, same sample spiked with Na₂SO₄ (the arrow corresponds to Na₂SO₄). For assay conditions, see Materials and Methods.