Kinetic assays to characterize fluorescent ligand binding to muscarinic acetylcholine M₁ receptor

Muscarinic acetylcholine receptors (M₁-M₅), which belong to the superfamily of G protein-coupled receptors (GPCRs), play a crucial role in regulating essential physiological processes and serve as significant targets in pharmaceutical research. The M₁ subtype is predominantly found within the central nervous system and is closely associated with the pathophysiology of neurodegenerative disorders. In recent times, fluorescent techniques have been frequently used to study ligand-receptor interactions. Among these methods, the fluorescence anisotropy (FA) assay stands out as a homogeneous assay for measuring ligand binding kinetics. In the scope of this study, we have implemented the FA method for M₁ receptor using fluorescent ligands UR-MK342 and UR-CG072, previously described to bind to muscarinic acetylcholine receptors. Additionally, we applied the same ligands in a kinetic fluorescence microscopy assay. Our findings revealed that the fluorescent ligand UR-MK342 had a slightly higher binding affinity and better dynamic range in both assays. We then used UR-MK342 to evaluate the apparent binding affinities (pK_i) of thirteen classical, three bitopic muscarinic acetylcholine receptor ligands, and three allosteric modulators and compared the obtained values to previously published literature. Notably, the online monitoring of competition binding experiments suggests a more complex mode of binding and UR-MK342 shows potential to be used in studies of allosteric modulation.