Abstract
β-lactamases are the foremost β-lactam resistance mechanism in Gram-negative bacteria. Class A β-lactamases collectively confer resistance to all available β-lactams. Carbapenems are a vital member of our β-lactam armamentarium, yet resistance against these agents has increased with the dissemination of carbapenem-hydrolysing class A β-lactamases. Moreover, co-administration of β-lactam antibiotics with a β-lactamase inhibitor (BLI) is often employed to circumvent β-lactamase mediated resistance, yet multiple inhibitor-resistant class A β-lactamases have emerged. This thesis combines experiment and computation to investigatethe carbapenem-hydrolysing activity of class A β-lactamases and their inhibition by BLIs, aiming to guide rational design of developments to current β-lactam scaffolds and novel BLIs. Molecular dynamics simulations of eight class A β-lactamases, including four carbapenemases, identify biochemical features that confer carbapenemase activity in class A β-lactamases. This is extended using QM/MM adaptive string method simulations of two tautomers of a carbapenem-derived KPC-2 acyl-enzyme, modelling the formation of the deacylation tetrahedral intermediate. These identified the Δ2 tautomer as the catalytically competent acyl-enzyme tautomer state. Furthermore, dynamical non-equilibrium molecular dynamics (D-NEMD) simulations identify networks that modulate class A β-lactamase activity
and highlight D-NEMD simulations as a potential predictive tool for protein design and allosteric inhibitor development. Additionally, characterisation of the previously
unstudied Vibrio diabolicus carbapenemase-1 (VDC-1) identified it as a novel class A
carbapenemase and identify the basis of potent inhibition of VDC-1 by the diazabicyclooctane (DBO) BLI avibactam. Finally, crystallographic analysis of the recently approved BLI enmetazobactam, and the related tazobactam, with the GES-1 β-lactamase show that acylated enmetazobactam, unlike tazobactam, makes electrostatic interactions with the α3α4 loop, potentially explaining the increased inhibitory activity of enmetazobactam, compared to tazobactam, against GES-1. Overall, this thesis explores the dynamic behaviour of class A β-lactamases, providing mechanistic insights into catalysis and inhibition, contributing to rational design pipelines of novel BLIs and β-lactams.
Date of Award | 4 Feb 2025 |
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Original language | English |
Awarding Institution |
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Supervisor | Jim Spencer (Supervisor) & Adrian J Mulholland (Supervisor) |