Electrostatic interactions influence diazabicyclooctane inhibitor potency against OXA-48-like β-lactamases

Joseph F. Hoff; Kirsty E. Goudar; Karina Calvopiña; Michael Beer; Philip Hinchliffe; John M. Shaw; Catherine L. Tooke; Yuiko Takebayashi; Andrew F. Cadzow; Nicholas J. Harmer; Adrian J. Mulholland; Christopher J. Schofield; James Spencer

doi:10.1039/d5md00512d

Electrostatic interactions influence diazabicyclooctane inhibitor potency against OXA-48-like β-lactamases

Joseph F. Hoff
, Kirsty E. Goudar
, Karina Calvopiña
, Michael Beer
, Philip Hinchliffe
, John M. Shaw
, Catherine L. Tooke
, Yuiko Takebayashi
, Andrew F. Cadzow
, Nicholas J. Harmer
, Adrian J. Mulholland
, Christopher J. Schofield
, James Spencer

University of Bristol
University of Oxford
University of Exeter

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

Abstract

Carbapenemases, β-lactamases hydrolysing carbapenem antibiotics, challenge the treatment of multi-drug resistant bacteria. The OXA-48 carbapenemase is widely disseminated in Enterobacterales, necessitating new treatments for producer strains. Diazabicyclooctane (DBO) inhibitors, including avibactam and nacubactam, act on a wide range of enzymes to overcome β-lactamase-mediated resistance. Here we describe investigations on how avibactam and nacubactam inhibit OXA-48 and two variants, OXA-163 and OXA-405, with deletions in the β5–β6 loop neighbouring the active site that modify activity towards different β-lactam classes. Nacubactam is ∼80-fold less potent than avibactam towards OXA-48, but this difference reduces in OXA-163 and OXA-405. Crystal structures and molecular dynamics simulations reveal electrostatic repulsion between Arg214 on the OXA-48 β5–β6 active-site loop and nacubactam, but not avibactam; effects absent from simulations of OXA-163 and OXA-405, which lack Arg214. Crystallographic and mass spectrometry data demonstrate that all three enzymes support desulfation of the bound DBOs. The results indicate that interactions with Arg214 affect DBO potency, suggesting that sequence variation in OXA-48-like β-lactamases affects reactivity towards inhibitors as well as β-lactam substrates.

Original language	English
Journal	RSC Medicinal Chemistry
Early online date	8 Aug 2025
DOIs	https://doi.org/10.1039/d5md00512d
Publication status	E-pub ahead of print - 8 Aug 2025

Data Availability Statement

Supplementary data (sequences of oligonucleotide primers; conditions, data processing and validation statistics for crystallographic experiments; steady-state kinetic parameters for hydrolysis of reporter substrate (nitrocefin); supplementary images of crystal structures; analyses of molecular simulations) are provided in the supplementary information (SI). See DOI: https://doi.org/10.1039/D5MD00512D. Structure factors and coordinates for all crystal structures presented here have been deposited with the Protein Data Bank (PDB) with the following accession numbers: uncomplexed OXA-48 (9H11), OXA-48 in complex with nacubactam (9H12), uncomplexed OXA-163 (9H13), OXA-163 in complex with avibactam (9H14), OXA-163 in complex with nacubactam (4 hour soak) (9H15), OXA-163 in complex with nacubactam (16 hour soak) (9HPV), uncomplexed OXA-405 (9H16), OXA-405 in complex with avibactam (9H17), OXA-405 in complex with nacubactam (9H18). Data from molecular dynamics simulations will be made freely available at the University of Bristol Research Data Repository (https://data.bris.ac.uk/). Analysis scripts will be made available upon request.

Acknowledgements

All simulations were conducted using the facilities of the Advanced Computing Research Centre at the University of Bristol (http://www.bris.ac.uk/acrc/). The authors thank Diamond Light Source (beamline I03, proposals 23269 and 31440) and the SOLEIL (beamline PROXIMA 2A) synchrotrons, and the respective beamline scientists, for their support collecting the X-ray diffraction data presented here. K. E. G., J. S. and C. J. S. acknowledge funding from the U.K. Biotechnology and Biological Sciences Research Council (BBSRC, BB/W001187/1). M. B. and A. F. C. were supported by the BBSRC-funded South West Biosciences Doctoral Training Partnership (BB/T008741/1). J. F. H. was supported by grant MR/W006308/1 for the GW4 BIOMED2 DTP, awarded to the Universities of Bath, Bristol, Cardiff and Exeter from the Medical Research Council (MRC)/UKRI. C. L. T., J. S. and A. J. M. thank the Medical Research Council for support through the grant MR/T016035/1. 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) to A. J. M. and J. S.

ASJC Scopus subject areas

Biochemistry
Molecular Medicine
Pharmacology
Pharmaceutical Science
Drug Discovery
Organic Chemistry

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Hoff, J. F., Goudar, K. E., Calvopiña, K., Beer, M., Hinchliffe, P., Shaw, J. M., Tooke, C. L., Takebayashi, Y., Cadzow, A. F., Harmer, N. J., Mulholland, A. J., Schofield, C. J., & Spencer, J. (2025). Electrostatic interactions influence diazabicyclooctane inhibitor potency against OXA-48-like β-lactamases. RSC Medicinal Chemistry. Advance online publication. https://doi.org/10.1039/d5md00512d

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abstract = "Carbapenemases, β-lactamases hydrolysing carbapenem antibiotics, challenge the treatment of multi-drug resistant bacteria. The OXA-48 carbapenemase is widely disseminated in Enterobacterales, necessitating new treatments for producer strains. Diazabicyclooctane (DBO) inhibitors, including avibactam and nacubactam, act on a wide range of enzymes to overcome β-lactamase-mediated resistance. Here we describe investigations on how avibactam and nacubactam inhibit OXA-48 and two variants, OXA-163 and OXA-405, with deletions in the β5–β6 loop neighbouring the active site that modify activity towards different β-lactam classes. Nacubactam is ∼80-fold less potent than avibactam towards OXA-48, but this difference reduces in OXA-163 and OXA-405. Crystal structures and molecular dynamics simulations reveal electrostatic repulsion between Arg214 on the OXA-48 β5–β6 active-site loop and nacubactam, but not avibactam; effects absent from simulations of OXA-163 and OXA-405, which lack Arg214. Crystallographic and mass spectrometry data demonstrate that all three enzymes support desulfation of the bound DBOs. The results indicate that interactions with Arg214 affect DBO potency, suggesting that sequence variation in OXA-48-like β-lactamases affects reactivity towards inhibitors as well as β-lactam substrates.",

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AU - Hoff, Joseph F.

AU - Goudar, Kirsty E.

AU - Calvopiña, Karina

AU - Beer, Michael

AU - Hinchliffe, Philip

AU - Shaw, John M.

AU - Tooke, Catherine L.

AU - Takebayashi, Yuiko

AU - Cadzow, Andrew F.

AU - Harmer, Nicholas J.

AU - Mulholland, Adrian J.

AU - Schofield, Christopher J.

AU - Spencer, James

PY - 2025/8/8

Y1 - 2025/8/8

N2 - Carbapenemases, β-lactamases hydrolysing carbapenem antibiotics, challenge the treatment of multi-drug resistant bacteria. The OXA-48 carbapenemase is widely disseminated in Enterobacterales, necessitating new treatments for producer strains. Diazabicyclooctane (DBO) inhibitors, including avibactam and nacubactam, act on a wide range of enzymes to overcome β-lactamase-mediated resistance. Here we describe investigations on how avibactam and nacubactam inhibit OXA-48 and two variants, OXA-163 and OXA-405, with deletions in the β5–β6 loop neighbouring the active site that modify activity towards different β-lactam classes. Nacubactam is ∼80-fold less potent than avibactam towards OXA-48, but this difference reduces in OXA-163 and OXA-405. Crystal structures and molecular dynamics simulations reveal electrostatic repulsion between Arg214 on the OXA-48 β5–β6 active-site loop and nacubactam, but not avibactam; effects absent from simulations of OXA-163 and OXA-405, which lack Arg214. Crystallographic and mass spectrometry data demonstrate that all three enzymes support desulfation of the bound DBOs. The results indicate that interactions with Arg214 affect DBO potency, suggesting that sequence variation in OXA-48-like β-lactamases affects reactivity towards inhibitors as well as β-lactam substrates.

AB - Carbapenemases, β-lactamases hydrolysing carbapenem antibiotics, challenge the treatment of multi-drug resistant bacteria. The OXA-48 carbapenemase is widely disseminated in Enterobacterales, necessitating new treatments for producer strains. Diazabicyclooctane (DBO) inhibitors, including avibactam and nacubactam, act on a wide range of enzymes to overcome β-lactamase-mediated resistance. Here we describe investigations on how avibactam and nacubactam inhibit OXA-48 and two variants, OXA-163 and OXA-405, with deletions in the β5–β6 loop neighbouring the active site that modify activity towards different β-lactam classes. Nacubactam is ∼80-fold less potent than avibactam towards OXA-48, but this difference reduces in OXA-163 and OXA-405. Crystal structures and molecular dynamics simulations reveal electrostatic repulsion between Arg214 on the OXA-48 β5–β6 active-site loop and nacubactam, but not avibactam; effects absent from simulations of OXA-163 and OXA-405, which lack Arg214. Crystallographic and mass spectrometry data demonstrate that all three enzymes support desulfation of the bound DBOs. The results indicate that interactions with Arg214 affect DBO potency, suggesting that sequence variation in OXA-48-like β-lactamases affects reactivity towards inhibitors as well as β-lactam substrates.

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SN - 2632-8682

JO - RSC Medicinal Chemistry

JF - RSC Medicinal Chemistry

ER -

Electrostatic interactions influence diazabicyclooctane inhibitor potency against OXA-48-like β-lactamases

Abstract

Data Availability Statement

Acknowledgements

ASJC Scopus subject areas

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