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Antimicrobial Agents and Chemotherapy, September 2008, p. 3135-3143, Vol. 52, No. 9
0066-4804/08/$08.00+0 doi:10.1128/AAC.01677-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Inhibition of OXA-1 β-Lactamase by Penems
,
Christopher R. Bethel,1
Anne M. Distler,2
Mark W. Ruszczycky,3
Marianne P. Carey,3
Paul R. Carey,3
Andrea M. Hujer,1
Magda Taracila,1
Marion S. Helfand,1,3
Jodi M. Thomson,2
Matthew Kalp,3
Vernon E. Anderson,3
David A. Leonard,4
Kristine M. Hujer,1
Takao Abe,5
Aranapakam M. Venkatesan,5
Tarek S. Mansour,5 and
Robert A. Bonomo1,2*
Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio,1 Departments of Pharmacology,2 Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio,3 Grand Valley State College, Grand Valley, Michigan,4 Wyeth Research, Chemical and Screening Sciences, Pearl River, New York5
Received 28 December 2007/ Returned for modification 23 January 2008/ Accepted 6 June 2008
The partnering of a β-lactam with a β-lactamase inhibitor is a highly effective strategy that can be used to combat bacterial resistance to β-lactam antibiotics mediated by serine β-lactamases (EC 3.2.5.6). To this end, we tested two novel penem inhibitors against OXA-1, a class D β-lactamase that is resistant to inactivation by tazobactam. The Ki of each penem inhibitor for OXA-1 was in the nM range (Ki of penem 1, 45 ± 8 nM; Ki 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 β-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 β-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 1H 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 β-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 β-lactamases present in gram-negative bacteria.
* Corresponding author. Mailing address: Infectious Diseases Section, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-3800, ext. 4399. Fax: (216) 231-3482. E-mail: robert.bonomo{at}med.va.gov
Published ahead of print on 16 June 2008.
Supplemental material for this article may be found at http://aac.asm.org/.
Antimicrobial Agents and Chemotherapy, September 2008, p. 3135-3143, Vol. 52, No. 9
0066-4804/08/$08.00+0 doi:10.1128/AAC.01677-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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