Inhibition of metallo-beta-lactamases by a series of mercaptoacetic acid thiol ester derivatives

A series of mercaptoacetic acid thiol esters have been identified as metallo-beta-lactamase inhibitors. Electrospray mass spectrometry (ESMS) has shown that irreversible inhibition of the Bacillus cereus II metallo-beta-lactamase by SB214751, SB214752, and SB213079 was concomitant with a 90-Da increase in mass of the enzyme. Tryptic digestion of the B. cereus II inhibited with SB214751 illustrated that the peptide fragment, containing the only cysteine of the enzyme, had undergone a mass increment of 90 Da. It was further demonstrated that B. cereus II hydrolyzed this type of compound across the thiol ester bond to yield mercaptoacetic acid. Mercaptoacetic acid is the only molecular fragment common to SB214751, SB214752, and SB213079, and free mercaptoacetic acid does not bind covalently to B. cereus II. Therefore, it is concluded that these compounds inhibit B. cereus II by the mechanism-based delivery of mercaptoacetic acid, forming a disulfide linkage with the active sites cysteine (predicted mass shift = +90 Da) under the aerobic conditions of the assay. The different thiol esters examined had a broad range of potencies against the metallo-beta-lactamases tested. For example SB214751, SB214752, and SB213079 all had 50% inhibitory concentrations of < 10 and > 1,000 microM for the Stenotrophomonas maltophilia L-1 and Bacteroides fragilis CfiA enzymes, respectively. SB216968 was particularly active against the Aeromonas hydrophila CphA metallo-beta-lactamase and was found to be an uncompetitive inhibitor of this enzyme (Ki = 3.9 microM), whereas it exhibited irreversible inhibition of the L-1 enzyme. These observations with this series of compounds have revealed subtle differences between the active sites of different metallo-beta-lactamases. Finally, a novel application for isothermal titration calorimetry for assessing the zinc chelating activity of candidate inhibitors is also presented.