Inhibition of OXA-1 β-Lactamase by Penems

Abstract

The partnering of a ␤-lactam with a ␤-lactamase inhibitor is a highly effective strategy that can be used to combat bacterial resistance to ␤-lactam antibiotics mediated by serine ␤-lactamases (EC 3.2.5.6). To this end, we tested two novel penem inhibitors against OXA-1, a class D ␤-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 ␤-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 <900 s and the recovery of OXA-1 ␤-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 ␤-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 ␤-lactamases present in gramnegative bacteria.

Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

“…A major difference between penem 1 and meropenem compared to clavulanate, sulbactam, and tazobactam is the presence of a double bond between the C 2 and C 3 positions in the former compounds. As previously shown, this sp 2 configuration plays a major role in the binding/catalysis of penems (clavulanate included) and carbapenems (2,21,37,38). Sulbactam and tazobactam, which possess an sp 3 hybridized R 2 side chain, are positioned in a different manner in the active site.…”

“…Nitrocefin (NCF) was purchased from Becton Dickinson, and clavulanic acid was a kind gift from GlaxoSmithKline. [1]) and ␤-lactamase inhibitors (clavulanate [2], sulbactam [3], tazobactam [4], meropenem [5; with the R 2 side chain at the C 2 position], ertapenem [6], doripenem [7], and penem 1 [8]) used in this study. The C atom numbering system is shown for meropenem.…”

“…The turnover number (t n ) or the partitioning of the initial enzyme-inhibitor complex between hydrolysis and enzyme inactivation, k cat /k inact (partition ratio), was determined as previously reported (2,17,53).…”

“…In the case of penem 1, previous insights into the mechanism of inactivation coupled with the timed mass spectrometry and UV difference spectra suggest that penem 1 forms a linear imine species that undergoes 7-endo-trig cyclization to ultimately form a cyclic enamine, the 1,4-thiazepine derivative (appearance of a chromophore at 360 to 380 nm) (2,5). We previously studied this penem inhibitor against OXA-1, a monomeric class D ␤-lactamase (2), SHV-1, and the R244S variant of SHV-1 ␤-lactamase, and we found similar results (2,53). It is also possible that these UV difference spectra represent other chemical changes to the enzyme/ inhibitor complex (e.g., closure of the C 7 heterocycle ring of the penem).…”

The Different Inhibition Mechanisms of OXA-1 and OXA-24 β-Lactamases Are Determined by the Stability of Active Site Carboxylated Lysine

“…1). Bethel et al (20) in 2008 proposed that penems inactivate OXA-1 ␤-lactamase efficiently by forming an unusual acyl-enzyme complex. Here, our results show that penem inhibitors have a high affinity for both OXA-1 and OXA-24 enzymes.…”