Mechanistic basis for inhibition of the extended-spectrum β-lactamase GES-1 by enmetazobactam and tazobactam

Michael Beer; Philip Hinchliffe; Marko Hanževački; Christopher R Bethel; Catherine L Tooke; Marc W Van der Kamp; Krisztina M Papp-Wallace; Robert A Bonomo; Stuart Shapiro; Adrian J Mulholland; James Spencer

doi:10.1002/1873-3468.70155

Mechanistic basis for inhibition of the extended-spectrum β-lactamase GES-1 by enmetazobactam and tazobactam

Michael Beer, Philip Hinchliffe, Marko Hanževački, Christopher R Bethel, Catherine L Tooke, Marc W Van der Kamp, Krisztina M Papp-Wallace, Robert A Bonomo, Stuart Shapiro, Adrian J Mulholland, James Spencer

Research output: Contribution to journal › Article › peer-review

Abstract

β-Lactamase-catalysed hydrolysis is the primary form of β-lactam antibiotic resistance in Gram-negative bacteria. The penicillanic acid sulfone (PAS) enmetazobactam is thought to inhibit extended-spectrum β-lactamases (ESBLs) by fragmentation of an initial acyl-enzyme to form an active-site lysinoalanine cross link. We investigate interactions of enmetazobactam and its congener tazobactam with GES-1, an ESBL with structural features of carbapenem-hydrolysing β-lactamases. Crystal structures show different breakdown products of the two inhibitors covalently bound to the catalytic Ser70, assigned using quantum mechanics/molecular mechanics (QM/MM) calculations. We find no evidence for lysinoalanine formation, with mass spectrometry indicating active enzyme regeneration, behaviour previously observed for carbapenem-hydrolysing enzymes, but not ESBLs. This work establishes that PAS inhibitors interact with diverse β-lactamases by differing mechanisms, which should inform development of future compounds.

Original language	English
Pages (from-to)	3284-3300
Number of pages	17
Journal	FEBS Letters
Volume	599
Issue number	22
Early online date	13 Sept 2025
DOIs	https://doi.org/10.1002/1873-3468.70155
Publication status	Published - 24 Nov 2025

Data Availability Statement

Input ﬁles and ligand parameters for all QM/MM geometry optimisations and single point energy calculations are made available at the University of Bristol Research Data Repository (https://data.bris.ac.uk/).All crystal structures presented here have atomic coordinates and structure factors deposited to the Protein Data Bank (PDB; https://www.rcsb.org/) under accession codes 9ENV, 9ENW, 9ENX and 9ENY.

Acknowledgements

MB was supported by the BBSRC-funded South West Biosciences Doctoral Training Partnership[BB/T008741/1]. AJM and MH thank EPSRC for sup-port (EP/W013738/1). MWVK thanks BBSRC for funding (BB/M026280/1). This work was supported in part by funds and/or facilities provided by the Cleve-land Department of Veterans Affairs, Award Number1I01BX001974 (RAB), from the Biomedical Laboratory Research & Development Service of the VA Ofﬁce of Research and Development, and the Geriatric Research Education and Clinical Center VISN 10.The content is solely the authors’ responsibility and does not necessarily represent the ofﬁcial views of the NIH or the Department of Veterans Affairs. This work is part of a project that has received funding from the European Research Council under the European Horizon 2020 research and innovation program(PREDACTED Advanced Grant Agreement no.101021207) awarded to AJM and JS. The QM/MM,QM active site model and QM acyl-adduct only geometry optimisation calculations presented here were carried out using the computational facilities of the Advanced Computing Research Centre, University of Bristol (http://www.bris.ac.uk/acrc/). The authors thank Diamond Light Source (beamline I03, proposals23269 and 31440) and the beamline scientists for their assistance that enabled the X-ray diffraction data presented here to be collected. Allecra Therapeutics provided enmetazobactam samples used in this work. The authors also would like to thank members of the EPSRC project FEHybCat for useful discussions onQM/MM geometry optimisations and single point energy calculations in Py-ChemShell

Keywords

beta-Lactamases/chemistry
beta-Lactamase Inhibitors/pharmacology
Tazobactam/pharmacology
Catalytic Domain
Crystallography, X-Ray
Penicillanic Acid/analogs & derivatives
Models, Molecular

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10.1002/1873-3468.70155Licence: CC BY

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title = "Mechanistic basis for inhibition of the extended-spectrum β-lactamase GES-1 by enmetazobactam and tazobactam",

abstract = "β-Lactamase-catalysed hydrolysis is the primary form of β-lactam antibiotic resistance in Gram-negative bacteria. The penicillanic acid sulfone (PAS) enmetazobactam is thought to inhibit extended-spectrum β-lactamases (ESBLs) by fragmentation of an initial acyl-enzyme to form an active-site lysinoalanine cross link. We investigate interactions of enmetazobactam and its congener tazobactam with GES-1, an ESBL with structural features of carbapenem-hydrolysing β-lactamases. Crystal structures show different breakdown products of the two inhibitors covalently bound to the catalytic Ser70, assigned using quantum mechanics/molecular mechanics (QM/MM) calculations. We find no evidence for lysinoalanine formation, with mass spectrometry indicating active enzyme regeneration, behaviour previously observed for carbapenem-hydrolysing enzymes, but not ESBLs. This work establishes that PAS inhibitors interact with diverse β-lactamases by differing mechanisms, which should inform development of future compounds.",

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author = "Michael Beer and Philip Hinchliffe and Marko Han{\v z}eva{\v c}ki and Bethel, \{Christopher R\} and Tooke, \{Catherine L\} and \{Van der Kamp\}, \{Marc W\} and Papp-Wallace, \{Krisztina M\} and Bonomo, \{Robert A\} and Stuart Shapiro and Mulholland, \{Adrian J\} and James Spencer",

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doi = "10.1002/1873-3468.70155",

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AU - Beer, Michael

AU - Hinchliffe, Philip

AU - Hanževački, Marko

AU - Bethel, Christopher R

AU - Tooke, Catherine L

AU - Van der Kamp, Marc W

AU - Papp-Wallace, Krisztina M

AU - Bonomo, Robert A

AU - Shapiro, Stuart

AU - Mulholland, Adrian J

AU - Spencer, James

PY - 2025/11/24

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N2 - β-Lactamase-catalysed hydrolysis is the primary form of β-lactam antibiotic resistance in Gram-negative bacteria. The penicillanic acid sulfone (PAS) enmetazobactam is thought to inhibit extended-spectrum β-lactamases (ESBLs) by fragmentation of an initial acyl-enzyme to form an active-site lysinoalanine cross link. We investigate interactions of enmetazobactam and its congener tazobactam with GES-1, an ESBL with structural features of carbapenem-hydrolysing β-lactamases. Crystal structures show different breakdown products of the two inhibitors covalently bound to the catalytic Ser70, assigned using quantum mechanics/molecular mechanics (QM/MM) calculations. We find no evidence for lysinoalanine formation, with mass spectrometry indicating active enzyme regeneration, behaviour previously observed for carbapenem-hydrolysing enzymes, but not ESBLs. This work establishes that PAS inhibitors interact with diverse β-lactamases by differing mechanisms, which should inform development of future compounds.

AB - β-Lactamase-catalysed hydrolysis is the primary form of β-lactam antibiotic resistance in Gram-negative bacteria. The penicillanic acid sulfone (PAS) enmetazobactam is thought to inhibit extended-spectrum β-lactamases (ESBLs) by fragmentation of an initial acyl-enzyme to form an active-site lysinoalanine cross link. We investigate interactions of enmetazobactam and its congener tazobactam with GES-1, an ESBL with structural features of carbapenem-hydrolysing β-lactamases. Crystal structures show different breakdown products of the two inhibitors covalently bound to the catalytic Ser70, assigned using quantum mechanics/molecular mechanics (QM/MM) calculations. We find no evidence for lysinoalanine formation, with mass spectrometry indicating active enzyme regeneration, behaviour previously observed for carbapenem-hydrolysing enzymes, but not ESBLs. This work establishes that PAS inhibitors interact with diverse β-lactamases by differing mechanisms, which should inform development of future compounds.

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KW - beta-Lactamase Inhibitors/pharmacology

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KW - Catalytic Domain

KW - Crystallography, X-Ray

KW - Penicillanic Acid/analogs & derivatives

KW - Models, Molecular

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JF - FEBS Letters

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ER -

Mechanistic basis for inhibition of the extended-spectrum β-lactamase GES-1 by enmetazobactam and tazobactam

Abstract

Data Availability Statement

Acknowledgements

Keywords

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