Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M2 Receptor

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

doi:10.1021/acs.jmedchem.9b02172

Abstract

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.

Highlights

Fluorescence-based techniques have been increasingly used for studying ligand-receptor interactions, and there is a growing demand for suitable fluorescent receptor ligands.Compared with radiolabeled probes, fluorescent ligands are advantageous with respect to safety precautions and waste disposal
Selected fluorescent ligands were characterized by flow cytometry and high-content imaging based binding studies as well as by confocal microscopy to investigate their applicability as molecular tools
(10,41 12,41 14,41 16-18,41 20,41 22, 23, 27, 28, 30) were prepared from previously reported amine-functionalized precursor compounds (8,42 15,39 21,42 26,38 2939) and the pyrylium dye 9 as well as the indolinium-type cyanine dye succinimidyl esters 11, 13 and 19 (Scheme 1). The latter contain the same fluorophore core structure but differ with respect to attached sulfonic acid groups leading to different net charges of the dye

Summary

Introduction

Fluorescence-based techniques have been increasingly used for studying ligand-receptor interactions, and there is a growing demand for suitable fluorescent receptor ligands. Fluorescent ligands are advantageous with respect to safety precautions and waste disposal They are applicable to fluorescence microscopy and flow cytometry, routine techniques in many laboratories, as well as to various homogeneous assay systems requiring specialized multimode plate readers, such as high-content imaging based assays, or polarization based fluorescence anisotropy receptor binding studies.. [3H]6 and [3H]7, Figure 1B), exhibiting high M2R affinity, we conjugated two types of redemitting fluorophores to previously reported amine-functionalized dibenzodiazepinone derivatives.. [3H]6 and [3H]7, Figure 1B), exhibiting high M2R affinity, we conjugated two types of redemitting fluorophores to previously reported amine-functionalized dibenzodiazepinone derivatives.38,39 This approach yielded twelve fluorescent MR ligands, which were studied with 54 respect to their M1R-M5R affinities. Selected fluorescent ligands were characterized by flow cytometry and high-content imaging based binding studies as well as by confocal microscopy to investigate their applicability as molecular tools

Results and Discussion

A NH2 a AD

Conclusion