Probe dependence of allosteric enhancers on the binding affinity of adenosine A 1 -receptor agonists at rat and human A 1 -receptors measured using NanoBRET.

Publisher: Wiley Country of Publication: England NLM ID: 7502536 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1476-5381 (Electronic) Linking ISSN: 00071188 NLM ISO Abbreviation: Br J Pharmacol Subsets: MEDLINE

Publication: London : Wiley
Original Publication: London, Macmillian Journals Ltd.

Purinergic P1 Receptor Agonists/*pharmacology ; Receptor, Adenosine A1/*metabolism; Allosteric Regulation ; Animals ; HEK293 Cells ; Humans ; Ligands ; Purinergic P1 Receptor Agonists/chemistry ; Rats ; Receptor, Adenosine A1/chemistry ; Receptor, Adenosine A1/genetics

Background and Purpose: Adenosine is a local mediator that regulates a number of physiological and pathological processes via activation of adenosine A 1 -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 A 1 -receptor. Here, we have investigated the species and probe dependence of two allosteric modulators on the binding characteristics of fluorescent and nonfluorescent A 1 -receptor agonists.
Experimental Approach: A Nano-luciferase (Nluc) BRET (NanoBRET) methodology was used. This used N-terminal Nluc-tagged A 1 -receptors expressed in HEK293T cells in conjunction with both fluorescent A 1 -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 A 1 -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 A 1 -receptor agonists in a species-dependent manner that involved increases in B max and pK D .
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 A 1 -receptor in intact living cells. Furthermore, our studies suggest that VCP171 and PD 81,723 may switch a proportion of A 1 -receptors to an active agonist conformation (R*).
(© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

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PG/14/95/31248 United Kingdom BHF_ British Heart Foundation; MR/N020081/1 United Kingdom MRC_ Medical Research Council

0 (Ligands)
0 (Purinergic P1 Receptor Agonists)
0 (Receptor, Adenosine A1)

Date Created: 20190116 Date Completed: 20200424 Latest Revision: 20210109

20240104

PMC6433648

10.1111/bph.14575

30644086