Targeting SRPK1 with novel potent and selective small molecule inhibitors inhibits choroidal neovascularisation through modulating VEGF-A alternative splicing

Abstract

Purpose :
Purpose: Anti-angiogenic VEGF inhibitors are the standard of care for wAMD but must be administered by intraocular injection and non-specifically inhibit all VEGF-A isoforms. Pathological angiogenesis occurs when the balance between pro-angiogenic VEGF-A₁₆₅a and anti-angiogenic VEGF-A₁₆₅b is switched by differential splicing of VEGF-A mRNA to the proangiogenic form. Small molecule inhibitors were identified that target SRPK1, the kinase promoting VEGF-A₁₆₅a splicing, thereby selectively inhibiting pro-angiogenic VEGF-A without inhibiting endogenous, cytoprotective, antiangiogenic VEGF-A₁₆₅b. Here we describe novel SRPK1 inhibitors that specifically inhibit pro-angiogenic VEGF-A isoform-driven angiogenesis underlying wAMD.

Methods :
Methods: Novel compounds, synthesized based on the structure of SRPK1, were tested in kinase assays, immunoprecipitation, immunoblotting and immunofluorescent assays. Alternative splicing and VEGF-A isoform expression was evaluated by RT-PCR and ELISA. Selectivity was analysed using kinome screens, differential scanning fluorimetry and kinome immunoprecipitation. Compounds were evaluated for toxicity in vitro and hERG inhibition. Efficacy was evaluated by laser-CNV in vivo in C57/Bl6 mice.

Results :
Novel compounds selectively bind to SRPK1 and dose-dependently inhibit SRPK1 kinase activity (IC50s<10 nM), SRSF1 phosphorylation and nuclear localization. The balance of anti-angiogenic VEGF-A₁₆₅b:pro-angiogenic VEGF-A₁₆₅a levels was specifically increased at the mRNA and protein level. Novel compounds were identified that were highly specific for SRPK1 over closely related kinases and in kinome screens. These SRPK1 inhibitors were not toxic to RPE cells and had improved hERG IC₅₀s >1 mM. Novel compounds potently inhibited laser-CNV following eye drop administration in mice (EC50s<0.5µM, n=6-8, P<0.05, One-way ANOVA).

Conclusions :
We developed highly potent and selective SRPK1 inhibitors that specifically target pro-angiogenic VEGF-A-driven choroidal neovascularization following eye drop administration. These compounds potentially offer more specific, efficacious and safer therapeutics for patients with wAMD.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.