Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, can act in tumor-induced angiogenesis by binding to
specific receptors on the surface of endothelial cells. One such receptor, VEGFR-2/KDR, plays a key role in VEGF-induced angiogenesis.
Here, we expressed the catalytic domain of VEGFR-2 as a soluble active kinase using Bac-to-Bac expression system, and investigated
correlations between VEGFR-2 activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. We
used these data to establish a convenient, effective and non-radioactive ELISA screening technique for the identification and evaluation of
potential inhibitors for VEGFR-2 kinase. We screened 200 RTK target-based compounds and identified one (TKI-31) that potently inhibited
VEGFR-2 kinase activity (IC50=0.596 AM). Treatment of NIH3T3/KDR cells with TKI-31 blocked VEGF-induced phosphorylation of KDR in
a dose-dependent manner. Moreover, TKI-31 dose-dependently suppressed HUVEC tube formation. Thus, we herein report a novel, efficient
method for identifying VEGFR-2 kinase inhibitors and introduce one, TKI-31, that may prove to be a useful new angiogenesis inhibitor.