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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 screening model of geldanamycin (GA) -Hsp90α combined with high-throughput fluorescence polarization and to explore new geldanamycin derivatives using this model. Methods: Fluorescence polarization was used to detect the fluorescence polarization of Hsp90α protein by using the principle of fluorescence polarization in a homogeneous solution with geldanamycin labeled with fluorescein isothiocyanate (FITC) as a ligand. The affinity of neogeldanamycin derivatives to Hsp90α was evaluated in a competitive manner in order to find better geldanamycin derivatives. RESULTS: The geldanamycin-Hsp90α binding model with fluorescence polarization was successfully established with a Z-factor of 0.641. Among the samples of geldanamycin derivatives tested, the IC50 of inhibition of GA-Hsp90α binding by GA-APML and GA-AEPD were 82.98 and 90.06 nmol.L-1, respectively. Conclusion: The established geldanamycin-Hsp90α binding fluorescence polarization model basically meets the standard for high-throughput experiments. The designed geldanamycin derivatives still possess the same Hsp90α affinity as geldanamycin, demonstrating that it is possible to engineer geldanamycin at these sites.