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

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 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 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*).