Targeting metallo-carbapenemases via modulation of electronic properties of cephalosporins.

The global proliferation of metallo-carbapenemase-producing Enterobacteriaceae has created an unmet need for inhibitors of these enzymes. The rational design of metallo-carbapenemase inhibitors requires detailed knowledge of their catalytic mechanisms. Nine cephalosporins, structurally identical except for the systematic substitution of electron-donating and withdrawing groups in the para position of the styrylbenzene ring, were synthesized and utilized to probe the catalytic mechanism of New Delhi metallo-β-lactamase (NDM-1). Under steady-state conditions, K(m) values were all in the micromolar range (1.5-8.1 μM), whereas k(cat) values varied widely (17-220 s(-1)). There were large solvent deuterium isotope effects for all substrates under saturating conditions, suggesting a proton transfer is involved in the rate-limiting step. Pre-steady-state UV-visible scans demonstrated the formation of short-lived intermediates for all compounds. Hammett plots yielded reaction constants (ρ) of -0.34 ± 0.02 and -1.15 ± 0.08 for intermediate formation and breakdown, respectively. Temperature-dependence experiments yielded ΔG(‡) values that were consistent with the Hammett results. These results establish the commonality of the formation of an azanide intermediate in the NDM-1-catalysed hydrolysis of a range cephalosporins with differing electronic properties. This intermediate is a promising target for judiciously designed β-lactam antibiotics that are poor NDM-1 substrates and inhibitors with enhanced active-site residence times.