Thermodynamic Cycle Analysis and Inhibitor Design against Beta-Lactamase

Abstract

Beta-lactamases are the most widespread resistance mechanism to beta-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 nM inhibitor (compound 1) in complex with AmpC beta-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 carboxylate-amide hydrogen 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 structural bases of these energies, X-ray crystal structures of N289A/1 and N289A/2 were determined to 1.49 and 1.39 A, respectively. These structures suggest that no significant rearrangement occurs in the mutant versus the wild-type complexes with both compounds. The mutant enzymes L119A and L293A were made to investigate the interaction between a phenyl ring in 1 and these residues. Whereas deletion of the phenyl itself diminishes affinity by 5-fold, the double-mutant cycles suggest that this energy does not come through interaction with the leucines, despite the close contact in the structure. The energies of these interactions provide key information for the design of improved inhibitors against beta-lactamases. The high magnitude of the ion-dipole interaction between Asn289 and the carboxylate of 1 is consistent with the idea that ionic interactions can provide significant net affinity in inhibitor complexes.

The Deacylation Mechanism of AmpC β-Lactamase at Ultrahigh Resolution

“…This mechanism is consistent with substrate modification studies by Mobashery, which suggested that analogues lacking the equivalent nitrogen were trapped in the acyl adduct, as well as inhibition and structural studies that suggested that β-lactams with displaced nitrogens acted as inhibitors. 26, 31 We note that studies of alternative depsipeptides substrates are not easily reconciled with this hypothesis since these substrates lack the lactam nitrogen. 40 That said, analogous roles for the equivalent thiol in these alternative substrates cannot be excluded.…”

Re‐examining the role of Lys67 in class C β‐lactamase catalysis

“…Although small‐molecule solution experiments suggested that a Tyr150‐like phenolic group was negatively charged at pH 7,19 NMR studies on the class C β‐lactamase from Citrobacter freundii GN346 suggest that the pKa of Tyr150 is above pH 11 in the apo‐enzyme,20 disfavoring the tyrosinate hypothesis. The substrate‐assisted catalysis model is supported by the observations that certain substrate analogs lacking the equivalent nitrogen were trapped in the acyl complex and that β‐lactam compounds with displaced nitrogens acted as inhibitors 13, 21. Some experiments have also suggested that the pKa of the lactam nitrogen may be between pH 5 and 6, consistent with the proposed general base function 22.…”

Inhibition of the Class C β-Lactamase from Acinetobacter spp.: Insights into Effective Inhibitor Design

“…However, Asn289 is not a well conserved residue among class C β-lactamases. The K i of this cephalothin analog increases from 1 nM with the E.coli AmpC to 29 nM with the E. cloacae P99 AmpC which has a Ser289, suggesting that the amino acid at position 289 plays a role for the affinity of compound 5 (47, 58). …”

“…Further, the R 1 amide recognition site formed by the interaction of the R 1 carbonyl and Asn152 in the E. coli AmpC differs from our ADC: 5 representation, as this same side chain carbonyl hydrogen bonds with Lys67 (46). These consensus binding sites were compiled exclusively from crystal structures of the E. coli AmpC, and our kinetic data and modeling analyses indicate the ADC β-lactamase may interact differently with boronic acid inhibitors than other class C enzymes (15, 46, 58). Our molecular representations of ADC in complex with the inhibitors were useful for developing hypotheses; however, we remain cognizant of modeling limitations, such as the lack of active site flexibility and the removal of water molecules during the ligand docking protocol.…”

“…That the chiral cephalothin analog 5 can maintain low nM K i for several phylogenetically divergent AmpC β-lactamases reflects not only the potency of this inhibitor, but also what may be an important plasticity of AmpCs (26, 46, 58). The significant repositioning of the boronic acid derivative revealed in our ADC: 5 model may be an indication of this enzyme’s versatility, causing the β-lactamase: ligand interactions to have different molecular correlates.…”