Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells

Front Mol Biosci. 2023 Mar 16:10:1119157. doi: 10.3389/fmolb.2023.1119157. eCollection 2023.


Dopamine receptors are G-protein-coupled receptors that are connected to severe neurological disorders. The development of new ligands targeting these receptors enables gaining a deeper insight into the receptor functioning, including binding mechanisms, kinetics and oligomerization. Novel fluorescent probes allow the development of more efficient, cheaper, reliable and scalable high-throughput screening systems, which speeds up the drug development process. In this study, we used a novel Cy3B labelled commercially available fluorescent ligand CELT-419 for developing dopamine D3 receptor-ligand binding assays with fluorescence polarization and quantitative live cell epifluorescence microscopy. The fluorescence anisotropy assay using 384-well plates achieved Z' value of 0.71, which is suitable for high-throughput screening of ligand binding. The assay can also be used to determine the kinetics of both the fluorescent ligand as well as some reference unlabeled ligands. Furthermore, CELT-419 was also used with live HEK293-D3R cells in epifluorescence microscopy imaging for deep-learning-based ligand binding quantification. This makes CELT-419 quite a universal fluorescence probe which has the potential to be also used in more advanced microscopy techniques resulting in more comparable studies.

Keywords: G protein-coupled receptor (GPCR); budded baculoviruses; deep-learning based image analysis; dopamine D3 receptor (D3R); fluorescence polarization (FP); fluorescent microscopy; fluorescent probe; ligand binding kinetics.

Grants and funding

This study was supported by the Estonian Research Council grant (PSG230), by the European Regional Development Fund via the Enterprise Estonia Applied Research Programme 2021, and by the COST action CA 18133 ERNEST. TL was supported by the Ustus Agur stipend by Republic of Estonia Education and Youth Board and Estonian Association of Information Technology and Telecommunications. ES is grateful for the financial support from the Consellería de Cultura, Educación e Ordenación Universitaria of the Galician Government: (grant: ED431B 2020/43), Centro Singular de Investigación de Galicia accreditation 2019-2022 (ED431G 2019/03) and the European Regional Development Fund (ERDF).