荧光偏振法建立格尔德霉素-Hsp90α结合高通量筛选模型及其应用  

Development and application of fluorescence polarization assay for high throughput screening of geldanamycin-Hsp90α binding

作　　者：董飚[1] 陶佩珍[1] 李艳萍[1] 李玉环[1] 

机构地区：[1]中国医学科学院医药生物技术研究所,北京100050

出　　处：《中国新药杂志》2011年第5期447-450,共4页Chinese Journal of New Drugs

基　　金：国家自然科学基金(3077260);中央级公益性科研院所基本科研业务专项(IMBF200806)

摘　　要：目的:建立格尔德霉素(geldanamycin,GA)-Hsp90α结合高通量荧光偏振法筛选模型及应用此模型探查新的格尔德霉素衍生物。方法:本模型利用荧光偏振原理,在均相溶液中,以异硫氰酸荧光素(FITC)标记的格尔德霉素作为配基,与Hsp90α蛋白作用,采用荧光仪检测荧光偏振值。以竞争性结合方式评价新格尔德霉素衍生物对Hsp90α的亲和力,以期找出更好的格尔德霉素衍生物。结果:成功建立了荧光偏振原理的格尔德霉素-Hsp90α结合模型,其Z因子可达0.641。所测试的格尔德霉素衍生物样品中,GA-APML和GA-AEPD抑制GA-Hsp90α结合的IC50分别为82.98和90.06 nmol.L-1。结论:建立的格尔德霉素-Hsp90α结合荧光偏振法模型基本达到进行高通量实验所需的标准。所设计格尔德霉素衍生物仍具备与格尔德霉素相同的对Hsp90α亲和力,说明了在这些位点对格尔德霉素进行改造是可行的。Objective：To establish a high throughput screening,fluorescence polarization assay of binding between Hsp90α and geldanamycin,to identify novel geldanamycin derivatives.Methods：The assay was based on the fluorescence polarization theory by using fluorescein isothiocyanate（FITC） labeled geldanamycin（GA） for binding to Hsp90α in homogeneous solution.Affinity of novel geldanamycin derivatives for Hsp90α was measured by competitive assay.Results：The fluorescence polarization assay for Hsp90 using a fluorescent GA ligand was successfully established,of which Z factor value reached 0.641.Among the measured geldanamycin derivative samples,GA-APML and GA-AEPD caused inhibition effects on the binding of GA-Hsp90α with IC50 of 82.98 and 90.06 nmol·L-1,respectively.Conclusion：The fluorescence polarization assay for GA-Hsp binding basically meets the standard required by high throughput screening experiments.The designed geldanamycin derivatives still have same affinity to Hsp90α as the geldanamycin itself,which shows it is feasible to modify the structure of geldanamycin at these sites.

关 键 词：荧光偏振 HSP90Α 格尔德霉素衍生物 

分 类 号：R965.1[医药卫生—药理学]