Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells
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 fluoresce...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-03-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2023.1119157/full |
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| author | Maris-Johanna Tahk Tõnis Laasfeld Tõnis Laasfeld Elo Meriste Jose Brea Maria Isabel Loza Maria Majellaro Maria Majellaro Marialessandra Contino Eddy Sotelo Ago Rinken |
| author_facet | Maris-Johanna Tahk Tõnis Laasfeld Tõnis Laasfeld Elo Meriste Jose Brea Maria Isabel Loza Maria Majellaro Maria Majellaro Marialessandra Contino Eddy Sotelo Ago Rinken |
| author_sort | Maris-Johanna Tahk |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T00:14:07Z |
| format | Article |
| id | doaj.art-22db54a4222f4329a1d283cd834504c0 |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-04-10T00:14:07Z |
| publishDate | 2023-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-22db54a4222f4329a1d283cd834504c02023-03-16T06:30:48ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2023-03-011010.3389/fmolb.2023.11191571119157Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cellsMaris-Johanna Tahk0Tõnis Laasfeld1Tõnis Laasfeld2Elo Meriste3Jose Brea4Maria Isabel Loza5Maria Majellaro6Maria Majellaro7Marialessandra Contino8Eddy Sotelo9Ago Rinken10Institute of Chemistry, University of Tartu, Tartu, EstoniaInstitute of Chemistry, University of Tartu, Tartu, EstoniaDepartment of Computer Science, University of Tartu, Tartu, EstoniaInstitute of Chemistry, University of Tartu, Tartu, EstoniaCentro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CiMUS), Universidade de Santiago de Compostela, Santiago, SpainCentro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CiMUS), Universidade de Santiago de Compostela, Santiago, SpainCentro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Universidade de Santiago de Compostela, Santiago, SpainCeltarys Research S.L., Santiago, SpainDipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari Aldo Moro, Bari, ItalyCentro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Universidade de Santiago de Compostela, Santiago, SpainInstitute of Chemistry, University of Tartu, Tartu, EstoniaDopamine 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.https://www.frontiersin.org/articles/10.3389/fmolb.2023.1119157/fullG protein-coupled receptor (GPCR)dopamine D3 receptor (D3R)ligand binding kineticsfluorescent probedeep-learning based image analysisbudded baculoviruses |
| spellingShingle | Maris-Johanna Tahk Tõnis Laasfeld Tõnis Laasfeld Elo Meriste Jose Brea Maria Isabel Loza Maria Majellaro Maria Majellaro Marialessandra Contino Eddy Sotelo Ago Rinken Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells Frontiers in Molecular Biosciences G protein-coupled receptor (GPCR) dopamine D3 receptor (D3R) ligand binding kinetics fluorescent probe deep-learning based image analysis budded baculoviruses |
| title | Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells |
| title_full | Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells |
| title_fullStr | Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells |
| title_full_unstemmed | Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells |
| title_short | Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells |
| title_sort | fluorescence based hts compatible ligand binding assays for dopamine d3 receptors in baculovirus preparations and live cells |
| topic | G protein-coupled receptor (GPCR) dopamine D3 receptor (D3R) ligand binding kinetics fluorescent probe deep-learning based image analysis budded baculoviruses |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2023.1119157/full |
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