β-Lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) contribute significantly to the longevity of the β-lactam antibiotics used to treat serious infections. In the quest to design more potent compounds and to understand the mechanism of action of known inhibitors, 6β-(hydroxymethyl) penicillanic acid sulfone (6β-HM-sulfone) was tested against isolates expressing the class A TEM-1 β-lactamase and a clinically important variant of the AmpC cephalosporinase of Pseudomonas aeruginosa, PDC-3. The addition of the 6β-HM-sulfone inhibitor to ampicillin was highly effective. 6β-HM-sulfone inhibited TEM-1 with an IC 50 of 12 ± 2 nM and PDC-3 with an IC 50 of 180 ± 36 nM, and displayed lower partition ratios than commercial inhibitors, with partition ratios (k cat/k inact) equal to 174 for TEM-1 and 4 for PDC-3. Measured for 20 h, 6β-HM-sulfone demonstrated rapid, first-order inactivation kinetics with the extent of inactivation being related to the concentration of inhibitor for both TEM-1 and PDC-3. Using mass spectrometry to gain insight into the intermediates of inactivation of this inhibitor, 6β-HM-sulfone was found to form a major adduct of +247 ± 5 Da with TEM-1 and +245 ± 5 Da with PDC-3, suggesting that the covalently bound, hydrolytically stabilized acyl-enzyme has lost a molecule of water (HOH). Minor adducts of +88 ± 5 Da with TEM-1 and +85 ± 5 Da with PDC-3 revealed that fragmentation of the covalent adduct can result but appeared to occur slowly with both enzymes. 6β-HM-sulfone is an effective and versatile β-lactamase inhibitor of representative class A and C enzymes.
Bibliographical noteFunding Information:
We thank Dr. Jean-Marie Frère (Université de Liège, Belgium) for the purified TEM-1 β-lactamase used in parts of this study. KMP-W is supported by the Veterans Affairs Career Development Program. PRC was supported by the National Institutes of Health ( R01 GM54072 ). JDB is supported by grant N-0871 from the Robert A. Welch Foundation . RAB is supported by the Veterans Affairs Merit Review Program (RAB), the National Institutes of Health ( RO1 AI063517-01 ), and Geriatric Research Education and Clinical Centre VISN 10 . Pfizer also supported this work.
- β-Lactamase inhibitor