Investigating Interactions of Class C β-Lactamases with β-Lactam-Based Inhibitors

Abstract

Antimicrobial resistance (AMR) is a global concern due to increased use and misuse of antibiotics. It is projected to cause 8.22 million AMR-associated deaths by 2050. Bacteria have developed various resistance mechanisms against antibiotics, rendering them ineffective as treatment options and decreasing their efficacy. One major mechanism of resistance is the enzymatic inactivation of drugs, such as by β-lactamases, which hydrolyse the β-lactam ring in β-lactam antibiotics. Class C β-lactamases, one of the most prevalent β-lactamase classes, are commonly found in clinically significant pathogens like the Enterobacterales family. These enzymes confer resistance to penicillins and cephalosporins, significantly limiting treatment options and contributing to increased morbidity and mortality rates.

GC1 and CMY-2 – chromosomally-encoded and plasmid-mediated cephalosporinases, respectively – are commonly associated with community-acquired and healthcare-associated infections, highlighting their clinical significance. A tandem repeat in GC1 allows resistance to widely used oxyimino- and 3rd-generation cephalosporins. CMY-2, often found in multidrug-resistant strains, exhibits increased potential for dissemination due to its plasmid-mediated nature, responsible for global spread.

Recombinant GC1 and CMY-2 enzymes were produced for structural and kinetic studies and have been purified for crystallisation trials, with the aim of characterising their interactions with investigational β-lactams designed to generate long-lasting inhibitory complexes with diverse β-lactamases. Parallel kinetic experiments established turnover rates for enzyme-catalysed hydrolysis, as well as the potency and mechanism of potential inhibition. Liquid chromatography mass spectrometry was used to identify rearrangement and fragmentation products of candidate inhibitors to further elucidate enzyme-inhibitor interactions. These findings will aid in identifying novel approaches to mitigate β-lactam degradation by class C β-lactamases.

Date of Award	9 Dec 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Jim Spencer (Supervisor) & Matthew B Avison (Supervisor)