Targeting metallo-carbapenemases via modulation of electronic properties of cephalosporins

Abstract

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, Km values were all in the micromolar range (1.5–8.1 μM), whereas kcat 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.