Radiolabeled Dibenzodiazepinone-Type Antagonists Give Evidence of Dualsteric Binding at the M₂ Muscarinic Acetylcholine Receptor

The dualsteric ligand approach, aiming at ligands with improved subtype selectivity, has been increasingly applied to muscarinic receptors (MRs). In this article, we present the synthesis and characterization of a M₂R subtype-preferring radiolabeled dibenzodiazepinone-type antagonist ([³H]­UNSW-MK259, [³H]19) and its homodimeric analogue [³H]­UR-AP060 ([³H]33). Saturation binding studies at the M₂R, using the orthosteric antagonist atropine to determine unspecific binding, proved that the monomeric and the dimeric compound bind to the orthosteric binding site (apparent K_d: 0.87 and 0.31 nM, respectively). Various binding studies with [³H]19 and [³H]33 at the M₂R, for instance, saturation binding experiments in the presence of the allosteric MR modulators W84 (8) or LY2119620 (9) (Schild-like analysis) suggested a competitive mechanism between the allosteric modulator and the dibenzodiazepinone derivatives, and thus a dualsteric binding mode of both 19 and 33. This was consistent with the results of M₂R MD simulations (≥2 μs) performed with 19 and 33.