Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M2 Receptor

Abstract

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 M₂R affinities (pK_i (radioligand competition binding): 9.10–9.59). Binding studies at M₁ and M₃–M₅ receptors indicated a M₂R preference. Flow cytometric and high-content imaging saturation and competition binding (M₁R, M₂R, and M₄R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM₂R cells). Results from dissociation and saturation binding experiments (M₂R) in the presence of allosteric M₂R 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 M₂R in imaging and binding studies and suggest that they have a dualsteric mode of action.