Tahk, Maris-Johanna and Torp, Jane and Ali, Mohammed A. S. and Fishman, Dmytro and Parts, Leopold and Grätz, Lukas and Mueller, Christoph and Keller, Max and Veiksina, Santa and Laasfeld, Tönis and Rinken, Ago (2022) Live-cell microscopy or fluorescence anisotropy with budded baculoviruses-which way to go with measuring ligand binding to M-4 muscarinic receptors? OPEN BIOLOGY, 12 (6): 220019. ISSN 2046-2441
Full text not available from this repository. (Request a copy)Abstract
M-4 muscarinic acetylcholine receptor is a G protein-coupled receptor (GPCR) that has been associated with alcohol and cocaine abuse, Alzheimer's disease, and schizophrenia which makes it an interesting drug target. For many GPCRs, the high-affinity fluorescence ligands have expanded the options for high-throughput screening of drug candidates and serve as useful tools in fundamental receptor research. Here, we explored two TAMRA-labelled fluorescence ligands, UR-MK342 and UR-CG072, for development of assays for studying ligand-binding properties to M-4 receptor. Using budded baculovirus particles as M-4 receptor preparation and fluorescence anisotropy method, we measured the affinities and binding kinetics of both fluorescence ligands. Using the fluorescence ligands as reporter probes, the binding affinities of unlabelled ligands could be determined. Based on these results, we took a step towards a more natural system and developed a method using live CHO-K1-hM(4)R cells and automated fluorescence microscopy suitable for the routine determination of unlabelled ligand affinities. For quantitative image analysis, we developed random forest and deep learning-based pipelines for cell segmentation. The pipelines were integrated into the user-friendly open-source Aparecium software. Both image analysis methods were suitable for measuring fluorescence ligand saturation binding and kinetics as well as for screening binding affinities of unlabelled ligands.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PROTEIN-COUPLED RECEPTORS; PHARMACOLOGICAL CHARACTERIZATION; ACETYLCHOLINE-RECEPTORS; SUBTYPES; ASSAY; ANTAGONIST; M1; M4; SELECTIVITY; AFFINITY; fluorescence anisotropy; microscopy; G protein-coupled receptor; muscarinic acetylcholine M-4 receptor; fluorescent ligands; deep learning |
| Subjects: | 600 Technology > 615 Pharmacy |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 16 Feb 2024 10:57 |
| Last Modified: | 16 Feb 2024 10:57 |
| URI: | https://pred.uni-regensburg.de/id/eprint/58110 |
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