Biochemical and biophysical studies on the Escherichia coli AmpC β-Lactamase

Antimicrobial resistance is one of the major global health concerns of the 21st century. While β-lactam antibiotics remain the most important class of antibacterials, their clinical use is increasingly compromised by resistance, most importantly through β-lactamase catalysed hydrolysis. More than 4000 different β-lactamases have been identified, which can collectively inactivate all clinically used β-lactam antibiotics. One successful strategy to combat resistance through β-lactamases is a combination therapy consisting of a β-lactam antibiotic and a β-lactamase inhibitor. However, β-lactamases are evolving rapidly, both adapting to new β-lactam antibiotics, and becoming resistant to inhibition by established β-lactamase inhibitors. This work describes biochemical and biophysical studies on interactions of the Escherichia coli AmpC β-lactamase (AmpCEC) with new and established β-lactamase inhibitors as well as β-lactam antibiotics.
Studies on the inhibition of AmpCEC by the clinical candidate enmetazobactam as described in Chapter 2 lead to a re-evaluation of the mechanism by which not just AmpCEC, but serine β-lactamases (SBLs) from all classes, are inhibited both by enmetazobactam and by the structurally related and most widely clinically used β-lactamase inhibitors, i.e. sulbactam, tazobactam, and clavulanic acid. The results suggest that previously reported substantial scaffold fragmentation likely does not (at least generally) contribute to transient β-lactamase inhibition by these inhibitors and reveal caveats on the interpretation of widely used mass spectrometric techniques to study SBL inhibition through covalent modification. Further they demonstrate scope for further development of penam sulfones and clavams for irreversible SBL inhibition. In Chapter 3 studies on AmpCEC inhibition by the clinically relevant diazabicyclooctanes (DBOs) avibactam, relebactam, nacubactam, and zidebactam are described. The results reveal significantly enhanced potency of zidebactam compared to the other DBOs, resulting from improved stabilisation of the tetrahedral transition state involved in generation of the covalent enzyme-DBO complex. However, the results also show potential for further DBO optimisation for AmpCEC inhibition by C2 acyl side chain optimisation. Chapter 4 focuses on AmpCEC inhibition by (bi)cyclic boronates, comparing taniborbactam, the first clinically approved dual-action inhibitor, acting on both SBLs and the mechanistically distinct metallo β-lactamases (MBLs), to structurally related compounds CB2 and CB3. Unexpectedly, next to the bicyclic taniborbactam bound to the nucleophilic serine of AmpCEC, structural studies on AmpCEC interaction with taniborbactam reveal a second taniborbactam molecule bound non-covalently at the AmpCEC surface, apparently in a tricyclic form. Structural analysis reveals that this tricyclic form is likely not accommodated at SBL active sites, but may contribute to inhibition of some MBLs, thus explaining the unusually broad spectrum of β-lactamase inhibition by taniborbactam. Preliminary structural studies on AmpCEC interactions with β-lactam antibiotics, as described in Chapter 5, demonstrate that apparently subtle changes both in the core motifs and the sidechains of the β-lactam antibiotics can have a drastic impact on the conformation of the β-lactam derived acyl-enzyme species with potential implications for their hydrolytic stability. Chapter 6 describes initial steps to study AmpCEC catalysis and inhibition through time-resolved serial crystallography. A micro-crystallisation protocol is developed for AmpCEC and used to study AmpCEC interaction with inhibitors at room temperature in the 100 ms – 30 min timeframe.
Overall, the work presented gives new insight into the molecular basis of AmpCEC inhibition by all currently clinically relevant classes of inhibitors, as well as AmpCEC conferred antimicrobial resistance. The results provide guidance for the further development of β-lactamase resistant antimicrobials and improved β-lactamase inhibitors to counteract antimicrobial resistance.

Keywords: enzyme kinetics
AMR
AmpC
antimicrobial resistance
β-lactamase
mechanism-based inhibitors
time-resolved serial crystallography
mass spectrometry
X-ray crystallography
Advisor: Brem, J
Schofield, C