Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M 2 Receptor

Fluorescently labeled dibenzodiazepinone-type muscarinic acetylcholine receptor (MR) antagonists, including dimeric ligands, were prepared using red-emitting cyanine dyes. Probes containing a fluorophore with negative charge showed high M R affinities (p (radioligand competition binding): 9.10-9.59). Binding studies at M and M -M receptors indicated a M R preference. Flow cytometric and high-content imaging saturation and competition binding (M R, M R, and M R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM R cells). Results from dissociation and saturation binding experiments (M R) in the presence of allosteric M R modulators (dissociation: W84, LY2119620, and alcuronium; saturation binding: W84) were consistent with a competitive mode of action between the fluorescent probes and the allosteric ligands. Taken together, these lines of evidence indicate that these ligands are useful fluorescent molecular tools to label the M R in imaging and binding studies and suggest that they have a dualsteric mode of action.