Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A₁ receptor ligands

Abstract

Background and purpose:The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties of fluorescent agonists and antagonists.Experimental approach:Chinese hamster ovary cells stably expressing the human adenosine A1 receptor and a cyclic 3′,5′ adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with whole cell [3H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5′ (N-ethylcarboxamido) adenosine (NECA).Key results:Derivatives of NECA and XAC with different fluorophores, but equivalent linker length, showed significant differences in their binding properties to the adenosine A1 receptor. The BODIPY 630/650 derivatives had the highest affinity. Linker length also affected the pharmacological properties, depending on the fluorophore used. Particularly in fluorescent agonists, higher agonist potency could be achieved with large or small linkers for dansyl and BODIPY 630/650 derivatives, respectively.Conclusions and implications:The pharmacology of a fluorescent ligand was critically influenced by both the fluorophore and the associated linker. Furthermore, our data strongly suggest that the physicochemical properties of the fluorophore/linker pairing determine where in the environment of the target receptor the fluorophore is placed, and this, together with the environmental sensitivity of the resulting fluorescence, may finally decide its utility as a fluorescent probe.This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x

Probing the pharmacology of G protein-coupled receptors with fluorescent ligands

“…For small molecule ligands, however, a linker is generally required to separate the pharmacophore from the fluorophore to allow access of the pharmacophore to the transmembrane orthosteric binding site. The composition and length of this linker can have major effects on both the pharmacological, physicochemical and photophysical properties of the resulting fluorescent ligands (Baker et al, 2010(Baker et al, , 2011Vernall et al, Fig. 1.…”

“…In addition, these properties are also heavily influenced by the choice of fluorophore which can change fluorescent ligand affinity, photophysics and lipid solubility (Baker et al, 2010;Vernall et al, 2013). For these reasons, each new fluorescent ligand, both peptide and small molecule, must be considered as a completely new pharmacological entity compared to the parent ligand and be fully characterised before use in further studies.…”

Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

“…Design of fluorescent ligands requires the knowledge of both the ligand and the pharmacophore (Leopoldo et al, 2009;Briddon et al, 2011). The various variables of this interaction have recently been studied in a very systematic way for ligands at the adenosine A 1 receptor (Baker et al, 2010).…”

Antagonist Selective Modulation of Adenosine A1 and A3 Receptor Pharmacology by the Food Dye Brilliant Black BN: Evidence for Allosteric Interactions

“…8B). Immediately after the addition of Brilliant Black BN, membrane fluorescence was still detectable, indicating that membrane binding of XAC-X-BY630 to adenosine receptors at the cell surface can still be monitored by virtue of the fact that the lipophilic BODIPY dye is in a membrane environment and protected from the quenching effect of Brilliant Black BN in the incubation medium (Baker et al, 2010). Within 2 min of infinite dilution in the presence of 50 M Brilliant Black BN, the membrane-bound fluorescent intensity was decreased by approximately 70% (Fig.…”