A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties
| Autor: | David O. Bates, Eleanor Star, Sebastian Oltean, Steve J Harper, Christopher W.J. Smith, Monica Lamici Ayine, Ling Li, Clare Gooding, Megan Stevens |
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| Přispěvatelé: | Smith, Christopher WJ [0000-0002-2753-3398], Bates, David O [0000-0003-4850-2360], Oltean, Sebastian [0000-0001-7890-8439], Apollo - University of Cambridge Repository, Smith, Christopher W. J. [0000-0002-2753-3398], Bates, David O. [0000-0003-4850-2360], Smith, Christopher W J [0000-0002-2753-3398] |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Cancer Research Protein family 38/90 Drug development 13/106 32 Biomedical and Clinical Sciences 3101 Biochemistry and Cell Biology Green fluorescent protein 03 medical and health sciences Exon 13/1 0302 clinical medicine Genetics 631/67/1059/153 14/19 5 Development of treatments and therapeutic interventions Molecular Biology 14/35 RC254-282 38/109 Messenger RNA Chemistry Alternative splicing Intron High-throughput screening article Neoplasms. Tumors. Oncology. Including cancer and carcinogens 64/110 Cell biology 13/31 030104 developmental biology 5.1 Pharmaceuticals 030220 oncology & carcinogenesis FOS: Biological sciences RNA splicing 14/63 631/154/1435/2163 Minigene 31 Biological Sciences |
| Zdroj: | Oncogenesis, Vol 10, Iss 5, Pp 1-12 (2021) Oncogenesis |
| DOI: | 10.17863/cam.70915 |
| Popis: | Funder: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC); doi: https://doi.org/10.13039/501100000268 Funder: Diabetes UK; doi: https://doi.org/10.13039/501100000361 Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3′ splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A’s terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3′ splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing. |
| Databáze: | OpenAIRE |
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