A new model for random screening inhibitors of vascular endothelial growth factor receptor 1 kinase.
OBJECTIVE
To establish a 96-well plate based kinase assay using a recombinant vascular endothelial growth factor (VEGF) receptor 1 kinase domain protein.


METHODS
A human VEGF receptor 1 kinase domain protein was expressed in E coli, and its activity was monitored by its ability of phosphorylating the polyE4Y substrate coated on the walls of 96-well plates with antibody recognition and a colorimetric readout. A random screening of a sample organic compound library was carried out, and the hits were characterized with a transformed cell line stably expressing VEGF receptor 1 protein.


RESULTS
An efficient E coli expression system for human VEGF receptor 1 kinase domain protein was constructed, and the purified recombinant protein was used to establish a practical screening assay for kinase inhibitors in vitro. Two thousand eight hundred organic compounds were screened, and two disubstituted furans (A1 and A5) with new structure showed inhibition of VEGF receptor 1 kinase. Compound A1 inhibited only phosphorylation of substrate, while compound A5 inhibited both autophosphorylation and substrate phosphorylation. Both inhibitors affected phosphorylation in the transformed cells.


CONCLUSIONS
The recombinant receptor kinase based assay is simple and effective in identifying kinase inhibitors.
Zhuang
Shu-Fei
SF
National Center for Drug Screening, Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Zhou
Cai-Hong
CH
Qian
Jing
J
Qian
Zhen
Z
Shibuya
Masabumi
M
Ye
Qi-Zhuang
QZ
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005663:Furans / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D011897:Random Allocation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014407:Tumor Cells, Cultured; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics
D020533:Angiogenesis Inhibitors; D004791:Enzyme Inhibitors; D005663:Furans; D011994:Recombinant Proteins; D040281:Vascular Endothelial Growth Factor Receptor-1