血管内皮生长因子受体-2抑制剂高通量筛选模型的建立和应用

Establishment and Application of High Throughput Screening Model for Kinase Insert Domain Receptor

目的利用均相时间分辨荧光(HTRF)技术建立血管内皮生长因子受体-2抑制剂高通量药物筛选模型,从化合物样品库中寻找小分子抑制剂。方法使用20μL检测体系,384低容量白板,采用均相时间分辨荧光技术对反应体系中的酶活性进行荧光检测,进而评价待测样品的抑制活性。模型建立的体系优化包括如下实验步骤:酶浓度及反应时间优化,ATP和底物米氏常数测定,质控实验包括反应体系信噪比实验、抑制剂SU5416的IC50测定,Z'因子的测定。在建立稳定模型检测体系之后,对本中心化合物库提供的10 560个样品进行活性筛选,并对部分活性化合物进行IC50的测定。结果在血管内皮生长因子受体-2活性体系优化实验中求得血管内皮生长因子受体-2激酶最适反应酶浓度为0.10 ng.μL-1,最适反应时间为15min,ATP Km=0.75μmol.L-1,底物Km=94.90 nmol.L-1,信噪比底物:SA=2∶1,Z'值为0.85,阳性药IC50=1.03μmol.L-1。通过部分初筛活性样品进行复筛研究,得到3个活性化合物S2-14,S2-16、S2-38 IC50分别为1.01×10-4,6.04×10-5,7.23×10-6mol.L-1。结论利用均相时间分辨荧光技术成功建立了血管内皮生长因子受体-2激酶高通量筛选模型,并进行了一定量的定向筛选研究,发现了若干先导化合物。本实验体系方法可靠,结果稳定,可作为抗血管新生天然产物筛选体系进行应用推广。

OBJECTIVE To establish a KDR high throughput screening model with homogeneous time resolved fluorescence （HTRF） detection technology in order to screen small molecular KDR inhibitors from the compound library. METHODS A 20 μL assay system in 384-well low-volume white microplate was developed with HTRF based fluorescence detection system to determine the enzyme activity. The optimization steps consisted of the following experiments ： enzyme concentration and incubation time optimiztion, ATP and substrate Km determination, quality control experiments including signal noise ratio inspection, the inhibitor SU5416 IC50 vali- dation, and Z＇ value calculation. After a stable assay system was accomplished, a HTS campaign was started with 10 560 samples from the compound library and the 1C50 of some compounds were determined. RESULTS The optimized KDR activity assay conditions were as follows： the kinase concentration was