Mechanistic studies of the inactivation of TEM-1 and P99 by NXL104, a novel non-beta-lactam beta-lactamase inhibitor

Abstract

NXL104 is a potent inhibitor of class A and C serine β-lactamases, including KPC carbapenemases. Native and NXL104-inhibited TEM-1 and P99 β-lactamases analyzed by liquid chromatography-electrospray ionization-time of flight mass spectrometry revealed that the inactivated enzymes formed a covalent adduct with NXL104. The principal inhibitory characteristics of NXL104 against TEM-1 and P99 β-lactamases were determined, including partition ratios, dissociation constants (K), rate constants for deactivation (k(2)), and reactivation rates. NXL104 is a potent inhibitor of TEM-1 and P99, characterized by high carbamylation efficiencies (k(2)/K of 3.7 × 10(5) M(-1) s(-1) for TEM-1 and 1 × 10(4) M(-1) s(-1) for P99) and slow decarbamylation. Complete loss of β-lactamase activity was obtained at a 1/1 enzyme/NXL104 ratio, with a k(3) value (rate constant for formation of product and free enzyme) close to zero for TEM-1 and P99. Fifty percent inhibitory concentrations (IC(50)s) were evaluated on selected β-lactamases, and NXL104 was shown to be a very potent inhibitor of class A and C β-lactamases. IC(50)s obtained with NXL104 (from 3 nM to 170 nM) were globally comparable on the β-lactamases CTX-M-15 and SHV-4 with those obtained with the comparators (clavulanate, tazobactam, and sulbactam) but were far lower on TEM-1, KPC-2, P99, and AmpC than those of the comparators. In-depth studies on TEM-1 and P99 demonstrated that NXL104 had a comparable or better affinity and inactivation rate than clavulanate and tazobactam and in all cases an improved stability of the covalent enzyme/inhibitor complex.

FIG. 1.

Molecular structures of β-lactamase inhibitors: SUL (a), CLA (b), TZB (d), NXL104 (d), and BLI-489 (e).

FIG. 2.

Mass spectra of native (a) and inhibited (c) TEM-1 β-lactamase and of native (b) and inhibited (d) P99 β-lactamase.

FIG. 3.

Inactivation of TEM-1 and P99 by NXL104 (a and c) and TZB (b and d) at various inhibitor/enzyme ratios after 5-min (□) or 30-min (▴) incubation. Each point of these graphs is the mean value obtained from at least two independent experiments.

FIG. 4.

Time course of TEM-1 (a) and P99 (b) reactivation following quasi-total enzyme inhibition by NXL104 (□), CLA (•), or TZB (▴). Typical curves are presented in the graphs. Each experiment was performed three times, and the shapes of the curves were similar in all cases.

FIG. 5.

Adduct formed between NXL104 and the active-site serine of β-lactamases.