On-cell saturation transfer difference NMR spectroscopy on ion channels: Characterizing negative allosteric modulator binding interactions of P2X7

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Monaco, Serena, Browne, Jacob, Wallace, Matthew, Angulo, Jesús and Stokes, Leanne (2025) On-cell saturation transfer difference NMR spectroscopy on ion channels: Characterizing negative allosteric modulator binding interactions of P2X7. Journal of the American Chemical Society, 147 (36). 32400–32411. ISSN 0002-7863

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Abstract

P2X7 receptors are important drug targets involved in pathologies ranging from psychiatric disorders to cancer. Being membrane embedded receptors, they are more challenging for structural characterization, and at present, we only have a small number of X-ray and cryo-EM structures for P2X7 bound to antagonists. We demonstrate that saturation transfer difference (STD) NMR on live mammalian cells (on-cell STD NMR) overexpressing P2X7 receptors allows further structural insight on the complexes of P2X7 with two potent negative allosteric modulators, namely, AZ10606120 and JNJ-47965567, via the determination of the binding epitope mapping of the interactions, e.g., the main region of contact between the ligand and the binding pocket. This approach, reported for the first time on membrane-embedded ion channels, in combination with molecular docking, allows us to propose the first NMR-validated ligand binding models for two antagonists as bound to human P2X7 receptors and to correlate the structural knowledge acquired with the pharmacology data. We highlight the transformative potential of this application to aid drug design efforts in a less resource-demanding fashion than X-ray crystallography and cryo-EM, and we envisage on-cell STD NMR to fast become an asset for structure–activity relationship studies helping knowledge-based development of efficient drugs targeting P2X7 and other ion channels/membrane-embedded proteins.

Item Type: Article
Additional Information: Funding information: S.M. acknowledges support from BBSRC (grant number BB/P010660/1). M.W. acknowledges support from UKRI Future Leaders Fellowship (grant number MR/T044020/1). L.S. and M.W. acknowledge support from UEA School of Pharmacy. J.A. acknowledges support from Ministerio de Ciencia e Innovación (grant AEI/10.13039/501100011033/PID2022-142879NB-I00) cofunded by the European Regional Development Fund (ERDF) “A way of making Europe” and BBSRC (grant number BB/P010660/1).
Uncontrolled Keywords: sdg 3 - good health and well-being ,/dk/atira/pure/sustainabledevelopmentgoals/good_health_and_well_being
Faculty \ School: Faculty of Science > School of Chemistry, Pharmacy and Pharmacology
UEA Research Groups: Faculty of Science > Research Groups > Molecular and Tissue Pharmacology
Faculty of Science > Research Groups > Pharmaceutical Materials and Soft Matter
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Depositing User: LivePure Connector
Date Deposited: 17 Sep 2025 09:30
Last Modified: 15 Oct 2025 00:14
URI: https://ueaeprints.uea.ac.uk/id/eprint/100419
DOI: 10.1021/jacs.5c02985

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