Design, Synthesis, and Evaluation of a New Fluorescent Ligand for the M2 Muscarinic Acetylcholine Receptor.

The M2 muscarinic acetylcholine receptor (M2R) is a G protein-coupled receptor involved in regulating cardiovascular functions and mediation of central muscarinic effects, such as movement, temperature control, and antinociceptive responses. Molecular probes targeting this receptor are therefore important in exploring its pathophysiological role at a molecular level. Herein, we report the design, synthesis, and evaluation of a new fluorescent probe for M2R based on an anthranilamide ligand. In radioligand binding experiments, the presented Oregon Green 488-labeled conjugate (33) exhibited high M2R affinity (K i = 2.4 nM), a moderate preference for the M2R over the M4 receptor, and excellent to pronounced M2R selectivity compared to the M1, M3, and M5 receptors. The utility of the probe was demonstrated in confocal, two-photon, and stimulated emission depletion nanoscopy (STED) imaging to specifically label the receptors in human embryonic kidney (HEK) 293T cells. These properties suggest that our probe may be utilized in advanced microscopy to study the pharmacology of the M2R.