Inhibition of OXA-1 beta-lactamase by penems

Abstract

The partnering of a beta-lactam with a beta-lactamase inhibitor is a highly effective strategy that can be used to combat bacterial resistance to beta-lactam antibiotics mediated by serine beta-lactamases (EC 3.2.5.6). To this end, we tested two novel penem inhibitors against OXA-1, a class D beta-lactamase that is resistant to inactivation by tazobactam. The K(i) of each penem inhibitor for OXA-1 was in the nM range (K(i) of penem 1, 45 +/- 8 nM; K(i) of penem 2, 12 +/- 2 nM). The first-order rate constant for enzyme and inhibitor complex inactivation of penems 1 and 2 for OXA-1 beta-lactamase were 0.13 +/- 0.01 s(-1) and 0.11 +/- 0.01 s(-1), respectively. By using an inhibitor-to-enzyme ratio of 1:1, 100% inactivation was achieved in <or=900 s and the recovery of OXA-1 beta-lactamase activity was not detected at 24 h. Covalent adducts of penems 1 and 2 (changes in molecular masses, +306 +/- 3 and +321 +/- 3 Da, respectively) were identified by electrospray ionization mass spectrometry (ESI-MS). After tryptic digestion of OXA-1 inactivated by penems 1 and 2, ESI-MS and matrix-assisted laser desorption ionization-time-of-flight MS identified the adducts of 306 +/- 3 and 321 +/- 3 Da attached to the peptide containing the active-site Ser67. The base hydrolysis of penem 2, monitored by serial (1)H nuclear magnetic resonance analysis, suggested that penem 2 formed a linear imine species that underwent 7-endo-trig cyclization to ultimately form a cyclic enamine, the 1,4-thiazepine derivative. Susceptibility testing demonstrated that the penem inhibitors at 4 mg/liter effectively restored susceptibility to piperacillin. Penem beta-lactamase inhibitors which demonstrate high affinities and which form long-lived acyl intermediates may prove to be extremely useful against the broad range of inhibitor-resistant serine beta-lactamases present in gram-negative bacteria.

FIG. 1.

Chemical structures of penem inhibitors (penem 1 and penem 2) and tazobactam (compound 3). Compound 4 is BRL 42715.

FIG. 2.

Determination of k_inact and K_i of tazobactam (a), penem 1 (b), and penem 2 (c) against OXA-1. Activity curves were obtained with an Agilent 8452 spectrophotometer and were analyzed by nonlinear least-squares fit of the data with the Origin (version 7.5) program.

FIG. 3.

Deconvoluted mass spectra were obtained on a Q-Star quadrupole time-of-flight mass spectrometer equipped with a nanospray source. (a) Deconvoluted spectrum of the OXA-1 β-lactamase; (b), deconvoluted spectrum of the OXA-1 β-lactamase inactivated with penem 1; (c) deconvoluted spectrum of the OXA-1 β-lactamase inhibited with penem 2. (d) Region of the deconvoluted mass spectrum of the OXA-1 β-lactamase tryptic digest. The sequence of the peptide at 5,038 amu is shown with the disulfide bond highlighted. This peptide spans from the N terminus at Ser18 to Lys56, with the disulfide bond being between Cys37 and Cys59. The active-site serine is located at Ser67 and is designated by an asterisk. (e) Region of the deconvoluted mass spectrum of the tryptic digest of OXA-1 β-lactamase inactivated with penem 1. The peak at 5,344 amu represents the covalent attachment of penem 1. The mass shift is indicated. (f) Region of the deconvoluted mass spectrum of tryptic digest of OXA-1 β-lactamase inactivated with penem 2. The peak at 5,359 amu is due to the covalent attachment of penem 2. The mass shift is indicated.

FIG. 4.

Penem 2 and the 1,4-thiazepine derivative of penem 2 were modeled in the active site of OXA-1 β-lactamase by using the Affinity and Docking modules of the Insight II program. (a) Connolly surface diagram of OXA-1 with penem 2 as a Henri-Michaelis complex; (b) Connolly surface diagram of OXA-1 with the 1,4-thiazepine derivative trapped in the binding pocket; (c) important H bonds from OXA-1 to the 1,4-thiazepine derivative.

FIG. 5.

Mechanism of inactivation of OXA-1 β-lactamases by penem 1.