Molecular basis of ligand binding and receptor activation at the human A3 adenosine receptor

Zhang, L.; Mobbs, J. I.; Bennetts, F. M.; Venugopal, H.; Nguyen, A. T. N.; Christopoulos, A.; van der Es, D.; Heitman, L. H.; May, L. T.; Glukhova, A.; Thal, D. M.

doi:10.1101/2025.01.12.632629

bioRxiv · 10.1101/2025.01.12.632629

Molecular basis of ligand binding and receptor activation at the human A3 adenosine receptor

Zhang, L.·Mobbs, J. I.·Bennetts, F. M.·Venugopal, H.·Nguyen, A. T. N.·Christopoulos, A.·van der Es, D.·Heitman, L. H.·May, L. T.·Glukhova, A.·Thal, D. M.

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Abstract

Adenosine receptors (ARs: A1AR, A2AAR, A2BAR, and A3AR) are crucial therapeutic targets, yet developing selective, efficacious drugs remains challenging. Here, we present high-resolution cryo-electron microscopy (cryo-EM) structures of the human A3AR in three distinct functional states: bound to the endogenous agonist adenosine, the clinically relevant agonist Piclidenoson, and the covalent antagonist LUF7602. These structures, complemented by mutagenesis and pharmacological studies, reveal a unique A3AR activation mechanism involving an extensive hydrogen bond network from the extracellular surface down to the orthosteric binding site. In addition, we identify a cryptic pocket that accommodates the N6-iodobenzyl group of Piclidenoson through a ligand-dependent conformational change of M1745.35. Our comprehensive structural and functional characterization of A3AR advances understanding of adenosine receptor pharmacology and establishes a foundation for developing more selective therapeutics for various disorders including inflammatory diseases, cancer, and glaucoma. TeaserStructures of the A3AR in different conformations reveal a unique activation mechanism and cryptic binding pocket.

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Zhang, L., Mobbs, J. I., Bennetts, F. M., Venugopal, H., Nguyen, A. T. N., Christopoulos, A., van der Es, D., Heitman, L. H., May, L. T., Glukhova, A., Thal, D. M.. 2025-01-15. Molecular basis of ligand binding and receptor activation at the human A3 adenosine receptor. https://doi.org/10.1101/2025.01.12.632629

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