ABSTRACT
BRET and fluorescence anisotropy (FA) are two fluorescence-based techniques used for the characterization of ligand binding to G protein-coupled receptors (GPCRs) and both allow monitoring of ligand binding in real time. In this study, we present the first direct comparison of BRET-based and FA-based binding assays using the human M2 muscarinic acetylcholine receptor (M2R) and two TAMRA (5-carboxytetramethylrhodamine)-labeled fluorescent ligands as a model system. The determined fluorescent ligand affinities from both assays were in good agreement with results obtained from radioligand competition binding experiments. The assays yielded real-time kinetic binding data revealing differences in the mechanism of binding for the investigated fluorescent probes. Furthermore, the investigation of various unlabeled M2R ligands yielded pharmacological profiles in accordance with earlier reported data. Taken together, this study showed that BRET- and FA-based binding assays represent valuable alternatives to radioactivity-based methods for screening purposes and for a precise characterization of binding kinetics supporting the exploration of binding mechanisms.
Subject(s)
Fluorescent Dyes/chemistry , Receptor, Muscarinic M2/metabolism , Rhodamines/chemistry , Animals , Bioluminescence Resonance Energy Transfer Techniques , CHO Cells , Cricetulus , Fluorescence Polarization , HEK293 Cells , Humans , Ligands , Sf9 CellsABSTRACT
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.
Subject(s)
Fluorescent Dyes/metabolism , Muscarinic Antagonists/metabolism , Phthalimides/metabolism , Quaternary Ammonium Compounds/metabolism , Receptor, Muscarinic M2/antagonists & inhibitors , Receptor, Muscarinic M2/metabolism , Animals , CHO Cells , Cholinergic Agents/chemistry , Cholinergic Agents/metabolism , Cholinergic Agents/pharmacology , Cricetulus , Fluorescent Dyes/chemistry , Muscarinic Antagonists/chemistry , Muscarinic Antagonists/pharmacology , Phthalimides/chemistry , Phthalimides/pharmacology , Protein Structure, Secondary , Quaternary Ammonium Compounds/chemistry , Quaternary Ammonium Compounds/pharmacologyABSTRACT
A series of fluorescent dibenzodiazepinone-type muscarinic acetylcholine M2 receptor (M2R) ligands was synthesized using various fluorescent dyes (5-TAMRA, λ ex/λ em ≈ 547/576 nm; BODIPY 630/650, λ ex/λ em ≈ 625/640 nm; pyridinium dye Py-1, λ ex/λ em ≈ 611/665 nm and pyridinium dye Py-5, λ ex/λ em ≈ 465/732 nm). All fluorescent probes exhibited high M2R affinity (pK i (radioligand competition binding): 8.75-9.62, pK d (flow cytometry): 8.36-9.19), a very low preference for the M2R over the M1 and M4 receptors and moderate to pronounced M2R selectivity compared to the M3 and M5 receptors. The presented fluorescent ligands are considered useful molecular tools for future studies using methods such as fluorescence anisotropy and BRET based MR binding assays.
ABSTRACT
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): p Ki (hM2R) of 8.97, ratio of Ki 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.
