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 (pK i (radioligand competition binding): 9.10-9.59). Binding studies at M 1 and M 3 -M 5 receptors indicated a M 2 R preference. Flow cytometric and high-content imaging saturation and competition binding (M 1 R, M 2 R and M 4 R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM 2 R cells). Results from dissociation and saturation binding experiments (M 2 R) in the presence of allosteric M 2 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 2 R in imaging and binding studies, and suggest that they have a dualsteric mode of action.

New fluorescent molecular tools for the muscarinic acetylcholine M2 receptor, bearing various fluorescent dyes, showed high M2 receptor affinity in flow cytometric saturation binding studies at CHO–hM2R cells (pKd > 8.3).

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Muscarinic acetylcholine receptors (MRs), comprising five subtypes (M1R–M5R) in humans, exhibit a high degree of structural similarity. Therefore, subtype-selective MR agonists and antagonists are lacking. We present an approach to highly M2R-selective MR antagonists based on the conjugation of di- or tripeptides to M2R-preferring dibenzodiazepinone-type MR antagonists. M2R selectivity was dependent on the peptide sequence and on the type of linker. The introduction of basic amino acids resulted in improved M2R selectivity (e.g., UR-AP148 (48): pK i (hM2R) of 8.97, ratio of K i M1R/M2R/M3R/M4R/M5R of 49:1:6500:60:400) compared to reported pyridobenzo- and dibenzodiazepinone-type MR ligands. A supposed dualsteric binding mode of the DIBA–peptide conjugates, such as 48, at MRs was supported by molecular dynamics simulations.