Selectivity of fluorescent ligands containing peptide-based linkers for the adenosine-A1 and – A3 receptors

Leigh Stoddart, Jessica Denman, Andrea Vernall, Barrie Kellam, Stephen Briddon, Stephen Hill. University of Nottingham, Nottingham, UK

Fluorescent ligands are being increasingly used to investigate aspects of G protein-coupled receptor (GPCR) pharmacology including dimerisation and membrane organisation. Fluorescent probes for GPCRs generally consist of a known orthosteric ligand attached to a fluorophore via a chemical linker. Previous studies have demonstrated that the linker is important in determining the affinity and efficacy of the resulting conjugates (Baker et al., 2010). Here, we examined whether introducing a charged water-soluble peptide linker into fluorescent adenosine receptor antagonist and agonists improved their properties relative to previously characterised compounds (Briddon et al., 2004).

As our reference compound, we used XAC-X-BY630 [2], a conjugate of the antagonist xanthine amine cogener (XAC) and BODIPY-630/650 through an aminohexanoyl linker (X). Four further fluorescent antagonists were synthesised and purified (to >99%) by RP-HPLC and their affinities at the adenosine-A1 and -A3 receptors (A1AR and A3AR) determined (Table 1). Affinities at the A1AR were assessed using a [3H]DPCPX competition binding assay and at A3AR using antagonist shifts of agonist concentration response curve in SPAP reporter gene assay as previously described (Baker et al., 2010).

Incorporation of a polyamide based linker (CA200645 (Stoddart et al., 2012) had little effect on A1AR affinity compared to XAC-X-BY630, but increased affinity at the A3AR. A number of dipeptide linkers were then examined. When compared to CA200645, the biggest increase in affinity at the A3AR was seen with a serine-tyrosine linker, although this had little effect on A1AR affinity. In contrast, an alanine-tyrosine linker, for example, retained a similar affinity to CA200645 at both receptors. Use of the green BODIPY-FL fluorophore in combination with a Ser-Tyr linker also showed favourable affinity at A3AR, and measurable A1AR affinity, where previously this fluorophore-ligand combination was inactive (Baker et al., 2010). Confocal imaging detected specific binding of XAC-ser-tyr-BY630 and XAC-ser-try-BYFL on the membrane of CHO cells expressing A1AR or A3AR.

Table 1 Affinity and potency values of fluorescent antagonists and agonist used in this study

Values represent mean±S.E.M. of 3-7 experiments conducted in triplicate.

A similar approach using NECA as the pharmacophore also generated a functional fluorescent agonist, when conjugated to BY630 via a tripeptide glycine-alanine-alanine linker. This agonist had a higher potency then NECA as determined by stimulation of A3AR internalisation (Table 1). Confocal images of CHO cells expressing A3AR-YFP showed distinct membrane-localised fluorescence of ligand following exposure to NECA-gly-ala-ala-BY630 (25nM, 5min, 22°C) which was blocked by pre-treatment with the antagonist, MRS1220 (100nM).

We have therefore demonstrated that use of peptide linkers generates functional fluorescent adenosine receptor agonists and antagonists of high potency. Further investigation of amino acid combinations will allow optimisation of selectivity and affinity across the adenosine receptor family.

Baker JG, Middleton R, Adams L, May LT, Briddon SJ, Kellam B, et al. Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A1 receptor ligands. (2010) Br J Pharmacol. 159(4):772-86

Briddon SJ, Middleton RJ, Cordeaux Y, Flavin FM, Weinstein JA, George et al.. (2004). Quantitative analysis of the formation and diffusion of A1-adenosine receptor-antagonist complexes in single living cells. Proc Natl Acad Sci USA 101(13):4673-8

Stoddart LA, Vernall AJ, Briddon SJ, Kellam B, Hill SJ.,. (2012) Fragment screening at adenosine-a(3) receptors in living cells using a fluorescence-based binding assay. Chem Biol; 19, 1105-1115

We thank the MRC for financial support