RT Journal Article
T1 Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells
A1 Tahk, Maris Johanna
A1 Laasfeld, Tõnis
A1 Meriste, Elo
A1 Brea Floriani, José Manuel
A1 Loza García, María Isabel
A1 Majellaro, Maria
A1 Contino, Marialessandra
A1 Sotelo, Eddy
A1 Rinken, Ago
K1 G protein-coupled receptor (GPCR)
K1 Dopamine D3 receptor (D3R)
K1 Ligand binding kinetics
K1 Fluorescent probe
K1 Deep-learning based image analysis
K1 Budded baculoviruses
K1 Fluorescent microscopy
K1 Fluorescence polarization (FP)
AB 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.
PB Frontiers Media
YR 2023
FD 2023-03-16
LK https://hdl.handle.net/10347/45352
UL https://hdl.handle.net/10347/45352
LA eng
NO Tahk, M.J., Laasfeld, T., Meriste, E., Brea, J., Loza, M.I., Majellaro, M., Contino, M., Sotelo, E. and Rinken, A. (2023). Fluorescence based HTS-compatible ligand binding assays for dopamine D 3 receptors in baculovirus preparations and live cells. Frontiers in Molecular Biosciences, 10. 10.3389/fmolb.2023.1119157
NO 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).
DS Minerva
RD 24 abr 2026