Termodynamic Cycle Analysis and Inhibitor Design Against beta-Lactamase

Beta-Lactamases are the most widespread resistance mechanism to ‚-lactam antibiotics, such as the penicillins and cephalosporins. Transition-state analogues that bind to the enzymes with nanomolar affinities have been introduced in an effort to reverse the resistance conferred by these enzymes. To understand the origins of this affinity, and to guide design of future inhibitors, double-mutant thermodynamic cycle experiments were undertaken. An unexpected hydrogen bond between the nonconserved Asn289 and a key inhibitor carboxylate was observed in the X-ray crystal structure of a 1 nMinhibitor (compound 1) in complex with AmpC ‚-lactamase. To investigate the energy of this hydrogen bond, the mutant enzyme N289A was made, as was an analogue of 1 that lacked the carboxylate (compound 2). The differential affinity of the four different protein and analogue complexes indicates that the carboxylateamidehydrogen bond contributes 1.7 kcal/mol to overall binding affinity. Synthesis of an analogue of 1 where the carboxylate was replaced with an aldehyde led to an inhibitor that lost all this hydrogen bond energy, consistent with the importance of the ionic nature of this hydrogen bond. To investigate the structuralbases of these energies, X-ray crystal structures of N289A/1 and N289A/2 were determined to 1.49 and 1.39 Å, respectively. These structures suggest that no significant rearrangement occurs in the mutant versus the wild-type complexes with both compounds.