Abstract
Background and PurposeAdenosine 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 ApproachA 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 ResultsPD 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 ImplicationsThese 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*).
Highlights
What is already knownAdenosine is a local reactive metabolite that has a major role in regulating a number of physiological and pathological processes including inflammation, hypoxia, and cardiovascular regulation (Fredholm, Ijzerman, Jacobson, Linden, & Müller, 2011)
VCP171 and, to a lesser extent, PD 81,723, increased the specific binding of three fluorescent A1‐receptor agonists in a species‐ dependent manner that involved increases in Bmax and pKD. 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
Our studies suggest that VCP171 and PD 81,723 may switch a proportion of A1‐receptors to an active agonist conformation (R*)
Summary
Adenosine is a local reactive metabolite that has a major role in regulating a number of physiological and pathological processes including inflammation, hypoxia, and cardiovascular regulation (Fredholm, Ijzerman, Jacobson, Linden, & Müller, 2011). Numerous selective agonists and antagonists for each adenosine receptor subtype are available for the study of receptor function (see Fredholm et al, 2011; Müller & Jacobson, 2011). 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. 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 (Hill, May, Kellam, & Woolard, 2014). These fluorescent ligands included the xanthine amine congener based antagonist ligand CA200645 (Corriden, Kilpatrick, Kellam, Briddon, & Hill, 2014; Stoddart, Johnstone, et al, 2015) and fluorescent A1‐agonists based on adenosine (ABA‐X‐BY630; Middleton et al, 2007; May, Self, Briddon, & Hill, 2010) or NECA (ABEA‐X‐BY630, Cordeaux, Briddon, Alexander, Kellam, & Hill, 2008; Stoddart, Johnstone, et al, 2015; BY630‐X‐(D)‐A‐(D)‐A‐G‐ABEA; Stoddart, Vernall, et al, 2015)
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