Click Chemistry-Enabled Conjugation Strategy for Producing Dibenzodiazepinone-Type Fluorescent Probes To Target M2 Acetylcholine Receptors

The development of fluorescently labeled receptor-targeting compounds represents a powerful pharmacological tool to study and characterize ligand-receptor interactions. Despite significant advances in developing sub-type-specific antagonists for muscarinic acetylcholine receptors (mAChRs), reports on antagonists feasible for Click Chemistry are less common. Here, we designed and synthesized an antagonist suitable for probe attachment through Click Chemistry, namely, dibenzodiazepinone (DIBA)-alkyne, based on a previously reported DIBA scaffold with a high binding affinity to type-2 mAChR (M₂R). To demonstrate the versatility of DIBA-alkyne as a building block for bioconjugates, we assembled DIBA-alkyne with Cyanine5 fluorophores (Cy5) and polyethylene glycol (PEG) biomolecules to obtain fluorescent DIBA antagonist (DIBA-Cy5) and fluorescent DIBA PEG derivatives. Flow cytometric analysis showed that DIBA-Cy5 possessed a high binding affinity to M₂R (K_d = 1.80 nM), a two-order magnitude higher binding affinity than M₁R. Fluorescent DIBA PEG derivatives maintained a potent binding to the M₂R (K_d ≤ 4 nM), confirmed by confocal microscopic imaging. Additionally, DIBA-Cy5 can serve as a fluorescent ligand in the receptor-ligand competitive binding assay for other mAChR ligands, an attractive alternative to the traditional radioligand-based assay. The competitive binding mode between DIBA-Cy5 and orthosteric antagonist atropine/allosteric modulator LY2119620 indicated a dualsteric binding mode of the DIBA-type antagonist to M₂R. Lastly, we demonstrated the direct staining of DIBA-Cy5 to M₂R receptors in the sinoatrial node of a mouse heart. The adaptability of the clickable DIBA antagonist to a wide range of fluorophores and biomolecules can facilitate its use in various biomedical applications such as binding assays that screen compounds for M₂R as the receptor target.