新方法随机筛选血管内皮生长因子受体1激酶的抑制剂

目的:利用重组血管内皮细胞生长因子(VEGF)受体1激酶蛋白质建立大规模随机筛选分子模型.方法:重组人源VEGF受体1激酶蛋白质的催化活性通过酶联免疫法检测96孔板上的底物磷酸化程度得到.用大规模随机筛选寻找抑制剂,并在稳定表达VEGF受体1的细胞系上研究它们的性质.结果:在研究VEGF受体1激酶蛋白质大肠杆菌表达体系的基础上,建立了大规模筛选模型.通过对2800个有机化合物的筛选,找到了两个二取代呋喃类的VEGF受体1激酶抑制剂(A1和A5).其中化合物A1能抑制底物磷酸化,而化合物A5则对激酶的自磷酸化和底物磷酸化都能抑制.同时A1和A5都能影响转染细胞上的受体磷酸化作用.结论:利用重组受体激酶建立的分子模型为寻找抗肿瘤血管生成抑制剂提供了一个简单而有效的方法.

A new model for random screening inhibitors of vascular endothelial growth factor receptor 1 kinase

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 ?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 (Al and A5) with new structure showed inhibition of VEGF receptor 1 kinase. Compound Al inhibited only phosphorylation of substrate, while compound A5 inhibited both autophosphorylation and substrate phosphorylation. Both inhibitors affected phosphorylation in the transformed cells. CONCLUSION; The recombinant receptor kinase based assay is simple and effective in identifying kinase inhibitors.