Molecular Target of a Novel Small Molecule Angiogenesis Inhibitor

(a) Objectives: Aim 1: To detect specific binding proteins of WAin human endothelial cells We plan to test the feasibility of employing WA as a cell permeable probe to detect its specific binding protein(s) from vascular endothelial cells. For this, (i) we will synthesize a biotin affinity analog of WA and validate the biological activity of biotinylated- WA by testing in endothelial cell proliferation, vascular sprouting, and NF-kappa B signal transduction assays. Subsequently, (ii) we will employ biotinylated- WA to perform binding assays and compare the pattern of biotinylated protein adducts across different types of human endothelial cells (e.g., human umbilical vein, choroid and dermis), using electrophoretic techniques with affinity blotting to detect binding targets of WA. Aim 2: To identify the biological target of WAf rom human endothelial cells We plan next to elaborate the use of biotinylated- WA as an affinity ligand to purify the human biological target(s) of WA from endothelial cells. For this, (i) we will use optimized binding conditions to purify protein adduct(s) of biotinylated- WA from human endothelial cells using avidin-sepharose chromatography for their isolation. To identify the WA-binding protein target(s), (ii) we will employ mass spectroscopy and peptide microsequencing techniques.