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Antimicrob. Agents Chemother. doi:10.1128/AAC.01677-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Inhibition of OXA-1 -Lactamase by Penems

Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH; Departments of Pharmacology, and Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH; Grand Valley State College, Grand Valley, MI; Wyeth Research, Chemical and Screening Sciences, Pearl River, NY

^* To whom correspondence should be addressed. Email: robert.bonomo{at}med.va.gov.

	Abstract

Partnering a -lactam with a -lactamase inhibitor is a highly effective strategy to combat bacterial resistance to -lactam antibiotics mediated by serine -lactamases (E.C.3.2.5.6). To this end, we tested 2 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 penem 1, 45 ± 8 nM; K_i penem 2, 12 ± 2 nM). The k_inact of penems 1 and 2 for OXA-1 -lactamase were 0.13 ± 0.01 s^-1 and 0.11 ± 0.01 s^-1, respectively. Using an Inhibitor (I) to Enzyme (E) ratio of 1:1, 100 % inactivation was achieved in 900 s and OXA-1 -lactamase activity recovery was not detected at 24 h. Covalent adducts of penems 1 and 2 ( +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 (MALDI) time of flight (TOF) identified 306 ± 3 and 321 ± 3 Da adducts attached to the peptide containing the active site Ser67. Base hydrolysis of penem 2 monitored by serial ¹H NMR 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/L effectively restored piperacillin susceptibility. Penem -lactamase inhibitors which demonstrate high affinity and form long-lived acyl intermediates may prove to be extremely useful against the broad range of inhibitor-resistant serine -lactamases present in Gram-negative bacteria.