Expression and purification of the catalytic domain of human vascular endothelial growth factor receptor 2 for inhibitor screening.
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 microM). 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.
Zhong
Li
L
Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang, Pu Dong, Shanghai 201203, PR China.
Guo
Xiao-Ning
XN
Zhang
Xiu-Hua
XH
Wu
Zhi-Xing
ZX
Luo
Xiao-Min
XM
Jiang
Hua-Liang
HL
Lin
Li-Ping
LP
Zhang
Xiong-Wen
XW
Ding
Jian
J
D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D002460:Cell Line; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D004791:Enzyme Inhibitors; D040301:Vascular Endothelial Growth Factor Receptor-2