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

Abstract

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*).

The effect of two selective A1‐receptor agonists and the bitopic ligand VCP746 on heart rate and regional vascular conductance in conscious rats

“…VCP171 certainly did not markedly enhance the contribution of A 1 ‐receptors in the final response, with the possible exception of a small effect on heart rate. VCP171 does not produce a large enhancement of the affinity of adenosine at the rat A 1 ‐receptor (Cooper et al, ) and it is likely that the positive A 1 ‐receptor allosteric action of the VCP171 entity within the larger VCP746 molecule is not sufficient to overcome the apparently more potent effect of the adenosine moiety on adenosine A 2A ‐ or A 2B ‐receptors in the cardiovascular system. This finding points to the need swap adenosine for a more A 1 ‐selective agonist within any future bitopic ligands and to match carefully the concentration range over which the orthosteric and allosteric components operate.…”

“…This finding points to the need swap adenosine for a more A 1 ‐selective agonist within any future bitopic ligands and to match carefully the concentration range over which the orthosteric and allosteric components operate. It is also worth pointing out that the probe dependence of allosteric modulators also needs to be considered (Cooper et al, ). For example, VCP171 does not produce a significant positive allosteric effect on the binding of capadenoson to the rat A 1 ‐receptor (Cooper et al, ).…”

“…One way in which the activity of endogenous adenosine can be subtly regulated at the level of its target receptor is via drugs that bind to an allosteric site on the receptor protein of interest. These allosteric modulators act to enhance or inhibit the binding of adenosine to its receptor binding site (the orthosteric site) and/or change the resulting functional response (Keov, Sexton, & Christopoulos, ; Hill et al, ; Göblyös & IJzerman, ; Kimatrai‐Salvador, Baraldi, & Romagnoli, ; Cooper et al, ). VCP171, (2‐amino‐4‐(3‐(trifluoromethyl)phenyl)thiophen‐3‐yl)(phenyl)methanone, has been described as a novel positive allosteric modulator for the adenosine A 1 ‐receptor (Aurelio et al, , ; Imlach, Bhola, May, Christopoulos, & Christie, ; Valant et al, ; Vincenzi et al, ).…”

Fluorescent ligands: Bringing light to emerging GPCR paradigms

“…While the allosteric modulators had little effect on the binding of the fluorescent antagonist, they enhanced the affinity of both fluorescent and non-fluorescent agonists. Interestingly, at the rat A 1 receptor, VCP171 increased the maximal binding capacity (B max ) with all three fluorescent agonists but only significantly increased the affinity of AAG-ABEA-X-BY630 (Cooper et al, 2019).…”

“…While the allosteric modulators had little effect on the binding of the fluorescent antagonist, they enhanced the affinity of both fluorescent and non‐fluorescent agonists. Interestingly, at the rat A 1 receptor, VCP171 increased the maximal binding capacity ( B max ) with all three fluorescent agonists but only significantly increased the affinity of AAG‐ABEA‐X‐BY630 (Cooper et al, ). This increase in agonist‐specific affinity suggested that the allosteric modulators were allowing a larger proportion of the receptor population to adopt a high‐affinity, R*, confirmation.…”