Conversion of a non-selective adenosine receptor antagonist into A3-selective high affinity fluorescent probes using peptide-based linkers

Abstract

Advances in fluorescence-based imaging technologies have helped propel the study of real-time biological readouts and analysis across many different areas. In particular the use of fluorescent ligands as chemical tools to study proteins such as G protein-coupled receptors (GPCRs) has received ongoing interest. Methods to improve the efficient chemical synthesis of fluorescent ligands remain of paramount importance to ensure this area of bioanalysis continues to advance. Here we report conversion of the non-selective GPCR adenosine receptor antagonist Xanthine Amine Congener into higher affinity and more receptor subtype-selective fluorescent antagonists. This was achieved through insertion and optimisation of a dipeptide linker between the adenosine receptor pharmacophore and the fluorophore. Fluorescent probe 27 containing BODIPY 630/650 (pK(D) = 9.12 ± 0.05 [hA3AR]), and BODIPY FL-containing 28 (pK(D) = 7.96 ± 0.09 [hA3AR]) demonstrated clear, displaceable membrane binding using fluorescent confocal microscopy. From in silico analysis of the docked ligand-receptor complexes of 27, we suggest regions of molecular interaction that could account for the observed selectivity of these peptide-linker based fluorescent conjugates. This general approach of converting a non-selective ligand to a selective biological tool could be applied to other ligands of interest.

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

“…Recent work in our laboratory has focused on the development of fluorescent ligands for the adenosine receptors with improved imaging properties. We found that inclusion of a peptidic linker in an XAC-BODIPY630/650 conjugate improved its affinity and significantly reduced the levels of intracellular non-specific binding (Vernall et al, 2013) compared to a direct XAC-BODIPY630/650 (XAC-X-BY630) conjugate ( Fig. 3) .…”

“…Using a fluorescent antagonist that emits at green wavelengths, the assay can be adapted to determine the affinity of fluorescent antagonists emitting at red wavelengths and vice versa (Vernall et al, 2013). Although the throughput of this system is not as high as for FRET or fluorescence polarisation assays (Allen et al, 2000;Veiksina et al, 2014;Zwier et al, 2010), one advantage of this imaging-based technique is the ability to simultaneously determine the affinity and efficacy of an unknown ligand and potentially to correlate this to what is happening at the single cell level.…”

“…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.…”

A Perspective on Studying G-Protein–Coupled Receptor Signaling with Resonance Energy Transfer Biosensors in Living Organisms

“…In addition, the ligand has to display the necessary receptor subtype selectivity to label the receptor of interest in a cell membrane that may contain other receptors for the same endogenous hormone or neurotransmitter. For example, most cells express multiple adenosine receptors, so it is important to develop highly selective fluorescent ligands for the specific adenosine receptor of interest (Vernall et al, 2013(Vernall et al, , 2014, especially in the context of an entire organism.…”

Synthesis of novel (benzimidazolyl)isoquinolinols and evaluation as adenosine A1 receptor tools

“…In an effort to develop a selective A 1 AR uorescent ligand we chose to avoid a xanthine amine congener (XAC)-based pharmacophore because of subtype selectivity concerns as a range of XACuorescent conjugates have been reported as either non AR subtype selective or A 3 AR selective. 7 Cosimelli et al recently reported a series of (benzimidazolyl)isoquinolines as selective antagonists for human A 1 AR (hA 1 AR), among them ligands 1-3 ( Fig. 1), 8 which were used as the inspiration for the uorescent ligand design reported herein.…”