Live-cell microscopy or fluorescence anisotropy with budded baculoviruses—which way to go with measuring ligand binding to M4 muscarinic receptors?

Open Biology

Dmytro Fishman¹  Mohammed A. S. Ali¹  Leopold Parts¹  Maris-Johanna Tahk²  Jane Torp²  Ago Rinken²  Santa Veiksina²  Tõnis Laasfeld²  Christoph Müller³  Max Keller³  Lukas Grätz³ 

[1] Department of Computer Science, University of Tartu, Narva Street 20, 51009 Tartu, Estonia;Institute of Chemistry, University of Tartu, Ravila 14a, 50411 Tartu, Estonia;Institute of Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany;

关键词: fluorescence anisotropy;    microscopy;    G protein-coupled receptor;    muscarinic acetylcholine M4 receptor;    fluorescent ligands;    deep learning;   

DOI  :  10.1098/rsob.220019

来源: DOAJ

【 摘 要 】

M4 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 M4 receptor. Using budded baculovirus particles as M4 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-hM4R 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.

【 授权许可】

Unknown