Novel SRPK1 inhibitors specifically target alternative splicing in human primary retinal epithelial cells

Abstract

PurposeSpecifically altering the splicing of VEGF‐A from pro‐angiogenic VEGF‐A165a to anti‐angiogenic VEGF‐A165b could prove effective in the treatment of wet AMD. VEGF‐A alternative splicing is controlled through RNA binding protein SRSF1 which when phosphorylated by the serine kinase SRPK1 induces pro‐angiogenic VEGF‐A165a expression. We aimed to use novel SRPK1 inhibitors that specifically alter splicing in retinal pigment epithelial (RPE) cells.MethodsRPE cells were treated with novel compounds, synthesized based on the structure of SRPK1, followed by extraction of RNA and protein. RT‐PCR and digital PCR was used to examine splicing of genes expressed in the eye or known targets of SRPK1, and ELISA and immunoblotting were used to detect VEGF‐A isoform expression.ResultsNovel SRPK1 inhibitors dose‐dependently increased the expression of the anti‐angiogenic VEGF‐A165b isoform. RT‐PCR showed that SRPK1 inhibition altered alternative splicing of MKNK2, a known splicing target of SRSF1, resulting in a 2 fold change to isoform a in RPE cells. No change in splicing was observed for BCL2L1, ARR1, CAMK2D, RAC1, FN1 or hnRNPB1/A2. Other SRSF1 targets such as TEAD1, BIM and MST1R were not expressed in RPE cells.ConclusionsWe have developed novel SRPK1 inhibitors that specifically target splicing within the retina and offer an alternative more specific therapeutics for patients with exudative AMD.