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

2020; American Chemical Society; Volume: 63; Issue: 8 Linguagem: Inglês

10.1021/acs.jmedchem.9b02172

1520-4804

Xueke She, Andrea Pegoli, Corinna G. Gruber, David Wifling, Jessica Carpenter, Harald Hübner, Mengya Chen, Jianfei Wan, Günther Bernhardt, Peter Gmeiner, Nicholas D. Holliday, Max Keller,

Pharmacological Receptor Mechanisms and Effects

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 (pKi (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.