title: Expression and purification of the catalytic domain of human vascular endothelial growth factor receptor 2 for inhibitor screening.
abstract: 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.
authors.affiliation: 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.
authors.forename: Li
authors.initials: L
authors.lastname: Zhong
authors.forename: Xiao-Ning
authors.initials: XN
authors.lastname: Guo
authors.forename: Xiu-Hua
authors.initials: XH
authors.lastname: Zhang
authors.forename: Zhi-Xing
authors.initials: ZX
authors.lastname: Wu
authors.forename: Xiao-Min
authors.initials: XM
authors.lastname: Luo
authors.forename: Hua-Liang
authors.initials: HL
authors.lastname: Jiang
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Lin
authors.forename: Xiong-Wen
authors.initials: XW
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Ding
mesh_terms: 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*
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D040301:Vascular Endothelial Growth Factor Receptor-2