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

## TL;DR

Scientists created a new fluorescent tool to study the M2 muscarinic acetylcholine receptor in cells using advanced imaging techniques.

## Contribution

A novel fluorescent ligand with high M2R affinity and selectivity was designed and validated for advanced microscopy.

## Key findings

- The Oregon Green 488-labeled anthranilamide ligand showed high M2R affinity (Ki = 2.4 nM).
- The probe demonstrated selectivity for M2R over M1, M3, M4, and M5 receptors.
- The ligand was successfully used in confocal, two-photon, and STED imaging to label M2R in HEK 293T cells.

## Abstract

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.