Avibactam is a covalent, reversible, non-β-lactam β-lactamase inhibitor

Abstract

Avibactam is a β-lactamase inhibitor that is in clinical development, combined with β-lactam partners, for the treatment of bacterial infections comprising gram-negative organisms. Avibactam is a structural class of inhibitor that does not contain a β-lactam core but maintains the capacity to covalently acylate its β-lactamase targets. Using the TEM-1 enzyme, we characterized avibactam inhibition by measuring the on-rate for acylation and the off-rate for deacylation. The deacylation off-rate was 0.045 min(-1), which allowed investigation of the deacylation route from TEM-1. Using NMR and MS, we showed that deacylation proceeds through regeneration of intact avibactam and not hydrolysis. Other than TEM-1, four additional clinically relevant β-lactamases were shown to release intact avibactam after being acylated. We showed that avibactam is a covalent, slowly reversible inhibitor, which is a unique mechanism of inhibition among β-lactamase inhibitors.

Fig. 1.

Structures of β-lactamase inhibitors used in this study.

Fig. 2.

k_obs vs. I plot for avibactam onset of inhibition vs. TEM-1. The ranges of avibactam concentrations were 60 nM to 2.5 μM in a stirred cuvette and 0.8 to 50 μM by stopped flow. Data were fit as described in Materials and Methods. Error bars shown are ±SEM from the fit to k_obs from the onset of inhibition time courses.

Fig. 3.

Recovery of activity time courses for β-lactamase inhibitors. Avibactam (AVI), tazobactam (TAZ) and clavulanic acid (CLA) were incubated with TEM-1 to allow acylation and then diluted to follow enzyme reactivation. Results shown are the averages of three measurements. Data for the avibactam time course were fit to Eq. 1 as described in Materials and Methods, yielding a k_off of 0.045 ± 0.022 min⁻¹ (mean ± 2 SD).

Fig. 4.

Hydrolysis of β-lactamase inhibitors. (A) ¹H Carr–Purcell–Meiboom–Gill NMR spectra of 40 μM tazobactam alone (Upper) and 40 μM tazobactam + 4 μM TEM-1 sampled after 5 min at 37 °C (Lower). (B) ¹H Carr–Purcell–Meiboom–Gill NMR spectra of 40 μM avibactam alone (overlay 1) and 40 μM avibactam + 4 μM TEM-1 sampled from 5 min to 24 h at 37 °C (overlays 2–5). Experiments were performed as described in SI Materials and Methods. Signals originating from the TEM-1 enzyme are labeled with asterisks.

Fig. 5.

Equilibration of avibactam-TEM-1 acyl-enzyme. (A) Mass spectra of acylated TEM-1 (EI*) after dilution to various concentrations and equilibration for 2 h at 37 °C. (B) Fit of measurement of equilibria between avibactam-TEM-1 acyl-enzyme complex and free avibactam + TEM-1 as a function of complex dilution. The percent avibactam bound was measured by TEM-1 protein MS (blue squares), avibactam MS (green triangles), and initial enzyme activity (red circles). For the avibactam MS titration, the observed % free avibactam was used to calculate the % bound by assuming a mass balance that fraction bound is equal to (1 − fraction unbound). Error bars shown are ±SEM from three measurements for each detection technique. For calculation of K_i*, data for the different detection methods were fit independently assuming the tight-binding condition (39). The value of K_i* determined by the three techniques is 2.1 ± 1.0 nM (mean ± 2 SD), and the solid line indicates the fit to this value.

Fig. 6.

Acyl-enzyme exchange between TEM-1 and CTX-M-15. (A) Time course of acyl-enzyme exchange from acyl-TEM-1 to apo-CTX-M-15 detected by MS. The peaks corresponding to apo- and acyl-enzyme forms are labeled. (B) Plot of apo- and acyl-enzyme species of TEM-1 and CTX-M-15 over time. (C) Acyl-enzyme exchange to apo-TEM-1 from donor-acylated CTX-M-15, KPC-2, E. cloacae P99, and P. aeruginosa AmpC. Time courses are shown in Fig. S5 and the percentages of acyl-enzyme species at the final time point for each reaction are depicted.,

Fig. 7.

Scheme for the inhibition of TEM-1 by avibactam.