Inhibition of OXA-1 beta-lactamase by penems.
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.
Bethel
Christopher R
CR
Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Research Service, Cleveland, Ohio 44106, USA.
Distler
Anne M
AM
Ruszczycky
Mark W
MW
Carey
Marianne P
MP
Carey
Paul R
PR
Hujer
Andrea M
AM
Taracila
Magda
M
Helfand
Marion S
MS
Thomson
Jodi M
JM
Kalp
Matthew
M
Anderson
Vernon E
VE
Leonard
David A
DA
Hujer
Kristine M
KM
Abe
Takao
T
Venkatesan
Aranapakam M
AM
Mansour
Tarek S
TS
Bonomo
Robert A
RA
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001643:Bridged Bicyclo Compounds / Q000737:chemistry / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D006571:Heterocyclic Compounds / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010878:Piperacillin / Q000494:pharmacology; D013329:Structure-Activity Relationship; D000078142:Tazobactam; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry
D000900:Anti-Bacterial Agents; D001643:Bridged Bicyclo Compounds; D004791:Enzyme Inhibitors; D006571:Heterocyclic Compounds; D065093:beta-Lactamase Inhibitors; D010397:Penicillanic Acid; C039404:beta-lactamase OXA-2; D001618:beta-Lactamases; D000078142:Tazobactam; D010878:Piperacillin