Thiomandelic acid, a broad spectrum inhibitor of zinc β-lactamases. Kinetic and spectroscopic studies

Resistance to β-lactam antibiotics mediated by metallo-β -lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid. A series of 35 analogues were synthesized and examined as metallo-β-lactamase inhibitors. The Ki values (Bacillus cereus enzyme) are 0.09 μM for R-thiomandelic acid and 1.28 μM for the S-isomer. Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together. Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-β-lactamases, with a submicromolar Ki value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented. The binding of thiomandelic acid to the B. cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg91. Amide chemical shift perturbations for residues 30-40 (the β 3-β4 loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors.