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Probe dependence of allosteric enhancers on the binding affinity of adenosine A1?receptor agonists at rat and human A1?receptors measured using NanoBRET


Samantha L. Cooper

Mark Soave


Peter J. Scammells

Professor of Cardiovascular Physiology and Pharmacology

Professor of Molecular Pharmacology


Background and Purpose: Adenosine is a local mediator that regulates a number of physiological and pathological processes via activation of adenosine A1?receptors. The activity of adenosine can be regulated at the level of its target receptor via drugs that bind to an allosteric site on the A1?receptor. Here, we have investigated the species and probe dependence of two allosteric modulators on the binding characteristics of fluorescent and nonfluorescent A1?receptor agonists.
Experimental Approach: A Nano?luciferase (Nluc) BRET (NanoBRET) methodology was used. This used N?terminal Nluc?tagged A1?receptors expressed in HEK293T cells in conjunction with both fluorescent A1?receptor agonists (adenosine and NECA analogues) and a fluorescent antagonist CA200645.
Key Results: PD 81,723 and VCP171 elicited positive allosteric effects on the binding affinity of orthosteric agonists at both the rat and human A1?receptors that showed clear probe dependence. Thus, the allosteric effect on the highly selective partial agonist capadenoson was much less marked than for the full agonists NECA, adenosine, and CCPA in both species. VCP171 and, to a lesser extent, PD 81,723, also increased the specific binding of three fluorescent A1?receptor agonists in a species?dependent manner that involved increases in Bmax and pKD.
Conclusions and Implications: These results demonstrate the power of the NanoBRET ligand?binding approach to study the effect of allosteric ligands on the binding of fluorescent agonists to the adenosine A1?receptor in intact living cells. Furthermore, our studies suggest that VCP171 and PD 81,723 may switch a proportion of A1?receptors to an active agonist conformation (R*).


Cooper, S. L., Soave, M., Jörg, M., Scammells, P. J., Woolard, J., & Hill, S. J. (2019). Probe dependence of allosteric enhancers on the binding affinity of adenosine A1‐receptor agonists at rat and human A1‐receptors measured using NanoBRET. British Journal of Pharmacology, 176(7), 864-878.

Journal Article Type Article
Acceptance Date Dec 4, 2018
Online Publication Date Mar 6, 2019
Publication Date 2019-04
Deposit Date Dec 19, 2018
Publicly Available Date Mar 25, 2019
Journal British Journal of Pharmacology
Print ISSN 0007-1188
Electronic ISSN 1476-5381
Publisher Wiley
Peer Reviewed Peer Reviewed
Volume 176
Issue 7
Pages 864-878
Keywords adenosine A1?receptor; allosteric modulator; allosterism; BRET; fluorescent ligand; ligand?binding
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