Development of an AlphaScreen-based high-throughput screening assay for inhibitors of human vascular endothelial growth factor receptor-3.

Vascular endothelial growth factor receptor 3 (VEGFR-3, also known as Flt4), the receptor for vascular endothelial growth factors (VEGFs) C and D, is expressed on lymphatic endothelium and plays an important role in lymphangiogenesis. Recent studies indicate that VEGFR-3 may be involved in tumor angiogenesis and growth, and so VEGFR-3 is an attractive target for researchers seeking to prevent tumor growth and metastasis. Hence we expressed and purified the CD (catalytic domain) of VEGFR-3 (VEGFR-3-CD) in Escherichia coli expression system and established a new simple and effective HTS (high-throughput screening) assay based on AlphaScreen strategy for VEGFR-3 inhibitors. Then we identified a novel cobalt complex that inhibited the phosphorylation of VEGFR-3 in a dose-dependent manner with an IC50 value of 338nM. This compound could be a useful new angiogenesis inhibitor, which would be investigated further for its therapeutic potentials.