Inactivation of a class A and a class C β-lactamase by 6β-(hydroxymethyl)penicillanic acid sulfone

beta-Lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) contribute significantly to the longevity of the beta-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, 6 beta-(hydroxymethyl)penicillanic acid sulfone (6 beta-HM-sulfone) was tested against isolates expressing the class A TEM-1 beta-lactamase and a clinically important variant of the AmpC cephalosporinase of Pseudomonas aeruginosa, PDC-3. The addition of the 6 beta-HM-sulfone inhibitor to ampicillin was highly effective. 6 beta-HM-sulfone inhibited TEM-1 with an IC50 of 12 +/- 2 nM and PDC-3 with an IC50 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, 6 beta-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, 6 beta-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 (H-O-H). 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. 6 beta-HM-sulfone is an effective and versatile beta-lactamase inhibitor of representative class A and C enzymes. Published by Elsevier Inc.