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

Abstract

AbstractM4 muscarinic receptor is a G protein-coupled receptor that has been associated with alcohol and cocaine abuse, Alzheimer’s disease and schizophrenia which makes it an interesting drug target. For many G protein-coupled receptors, the development of high-affinity fluorescence ligands has expanded the options for high throughput screening of drug candidates and serve as useful tools in fundamental receptor research. So far, the lack of suitable fluorescence ligands has limited studying M4 receptor ligand binding. Here, we explored the possibilities of using fluorescence-based methods for studying binding affinity and kinetics to M4 receptor of both labeled and unlabeled ligands. We used two TAMRA-labeled fluorescence ligands, UR-MK342 and UR-CG072, for assay development. Using budded baculovirus particles as M4 receptor preparation and fluorescence anisotropy method, we determined the affinities and binding kinetics of both fluorescence ligands. The fluorescence ligands could also be used as reported probes for determining binding affinities of a set of unlabeled ligands. Based on these results, we took a step further towards a more natural signaling system and developed a method using live CHO-K1-hM4R cells and automated fluorescence microscopy suitable for routine determination of unlabeled 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 developed methods were suitable for measuring fluorescence ligand saturation binding, association and dissociation kinetics as well as for screening binding affinities of unlabeled ligands.Abstract Figure