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

She, Xueke; Pegoli, Andrea; Gruber, Corinna G.; Wifling, David; Carpenter, Jessica; H�bner, Harald; Chen, Mengya; Wan, Jianfei; Bernhardt, G�nther; Gmeiner, Peter; Holliday, Nicholas D.; Keller, Max

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.

Citation

She, X., Pegoli, A., Gruber, C. G., Wifling, D., Carpenter, J., Hübner, H., Chen, M., Wan, J., Bernhardt, G., Gmeiner, P., Holliday, N. D., & Keller, M. (2020). Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M2 Receptor. Journal of Medicinal Chemistry, 63(8), 4133-4154. https://doi.org/10.1021/acs.jmedchem.9b02172

• Journal Article Type: Article
• Acceptance Date: Apr 1, 2020
• Online Publication Date: Apr 1, 2020
• Publication Date: Apr 23, 2020
• Deposit Date: Apr 13, 2020
• Publicly Available Date: Apr 2, 2021
• Journal: Journal of Medicinal Chemistry
• Print ISSN: 0022-2623
• Electronic ISSN: 1520-4804
• Publisher: American Chemical Society
• Peer Reviewed: Peer Reviewed
• Volume: 63
• Issue: 8
• Pages: 4133-4154
• DOI: https://doi.org/10.1021/acs.jmedchem.9b02172
• Keywords: Molecular Medicine; Drug Discovery
• Public URL: https://nottingham-repository.worktribe.com/output/4289616
• Publisher URL: https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b02172
• Additional Information: This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Medicinal Chemistry ,copyright ©American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see ; https://doi.org/10.1021/acs.jmedchem.9b02172