Development of new anti-VEGF drug by SRPK1 inhibitor screening

Purpose AMD is a major cause of blindness. Current therapy aims to suppress VEGF levels in order to prevent CNV, which causes AMD. Recently we found that SRPIN340, a specific inhibitor of SR protein kinase 1 (SRPK1) which phosphorylates Serine-arginine-rich (SR) proteins and regulates splicing and transcription, suppressed the expression of VEGF and CNV. Here we screened for more active SRPK1 inhibitors which suppressed CNV effectively than SRPIN340. Methods In-silico screening; Structure-based virtual screening (SBVS) was applied against the general chemical library, which include 71955 compounds. In vitro kinase assay; The reaction mixture was incubated at 30 °C for 15 min and detected the residual ATP was measured by Kinase Glow following the manufacturer’s instruction. VEGF ELISA; We added 10μM compounds or control to ARPE-19 cells. The medium was collected 72 hours after the addition of the compounds. We evaluated the VEGF protein level in the culture medium by ELISA with manufacturer’s instruction. In vivo examination; The compound was intravitreally injected to laser CNV model mice immediately after laser photocoagulation. Seven days after laser injury, the mice were perfused with 1mL of 0.5% FITC-labeled dextran. Flat mounts of RPE-choroid complex were obtained and the area of CNV was measured by fluorescence microscopy. Results 11994 compounds were selected by SBVS, and we were able to obtain 9535 compounds among them. Next we screened by in vitro kinase assay and selected four compunds, which were named SRPIN801-804. SRPIN(03 decreased the VEGF protein levels most effectively as measured by VEGF ELISA, and suppressed CNV growth in a dose-dependent manner. Conclusion We obtained a new anti-VEGF drug, which inhibits SRPK1 kinase acitivity.