Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A 1 Receptor in Living Cells.

The adenosine A ₁ receptor (A ₁ AR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of A ₁ AR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying A ₁ AR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of A ₁ AR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel A ₁ AR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of A ₁ AR molecular pharmacology and signaling in living cells.