FLK-1 AS A TARGET FOR TUMOR-GROWTH INHIBITION

A number of growth factor receptor tyrosine kinases have been implicat ed in angiogenesis, including epidermal growth factor receptor, fibrob last growth factor receptor, platelet-derived growth factor receptor, Flk-1/KDR, Flt-1, Tie-1, and Tek/Tie-2. Flk-1/KDR, a receptor for vasc ular endothelial growth factor (VEGF), is expressed exclusively in end othelial cells. Using dominant-negative methods, Flk-1 was shown to pl ay a role in angiogenesis and the growth of a variety of tumor types, Because of this, a drug discovery effort was established to identify F lk-1 kinase inhibitors. For initial screening, an ELISA in a 96-well f ormat was used to measure VEGF-induced Flk-1 tyrosine phosphorylation in whole cells, Compounds that inhibited ligand-induced receptor autop hosphorylation were confirmed by antiphosphotyrosine immunoblotting. I nhibition of VEGF-stimulated DNA synthesis in human endothelial cells was also assessed. Inhibitors were further evaluated for their effects on vessel formation using the chorioallantoic membrane assay. Using t hese methods, antiangiogenesis compounds that inhibit Flk-1 tyrosine k inase activity, endothelial cell mitogenesis, and blood vessel formati on in the chorioallantoic membrane assay have been found.