Reversibility of Covalent, Broad-Spectrum Serine β-Lactamase Inhibition by the Diazabicyclooctenone ETX2514.

Abstract

ETX2514 is a non-β-lactam serine β-lactamase inhibitor in clinical development that has greater potency and broader spectrum of β-lactamase inhibition than the related diazabicyclooctanone avibactam. Despite opening of its cyclic urea ring upon acylation, avibactam can recyclize and dissociate intact from certain β-lactamases. We investigated reversibility of ETX2514 acylation of 10 serine β-lactamases representing Ambler classes A, C, and D. Dissociation rate constants varied widely between enzymes and were lowest for class D. For most enzymes, the covalent adduct mass was that of ETX2514 (277 Da). OXA-10 was acylated with 277 and 197 Da adducts, consistent with loss of the sulfate moiety. KPC-2 showed only the 197 Da adduct. ETX2514 recyclized and dissociated intact from AmpC, CTX-M-15, P99, SHV-5 and TEM-1 but not from KPC-2, OXA-10, OXA-23, OXA-24, or OXA-48. Inactivation partition ratios were 1 for all enzymes except KPC-2, for which it increased to 3.0 after 2 h. This result and mass spectrometry showed that KPC-2 very slowly degraded ETX2514. Nevertheless, ETX2514 restored β-lactam activity to equal potency against isogenic Pseudomonas aeruginosa strains each overexpressing one of the 10 β-lactamases.