A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties

Abstract

Abstract Alternative splicing of the vascular endothelial growth factor A ( VEGF-A ) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-A xxx a 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-A xxx b 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-A xxx a and EGFP denotes VEGF-A xxx b). 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.

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