利用荧光极化技术在均相系统内建立基于Bcl-2/Bak作用模式的高通量药物筛选体系  

The construction of high throughput screening method based on Bcl-2/Bak interaction mode using fluorescence polarization technology in liquid system

作　　者：胡美茹[1] 靳宝锋[2] 张学敏[2] 孙瑛勋[1] 沈倍奋[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]军事医学科学院生物医学分析中心,北京100850

出　　处：《军事医学科学院院刊》2005年第5期457-459,470,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家高技术研究发展计划"863"资助项目(2001AA233041)

摘　　要：目的:利用荧光极化原理,在液相系统内建立基于Bcl-2/Bak相互作用模式的高通量药物筛选体系。方法:在液相系统内建立Bcl-2蛋白和多肽的相互作用体系,用光度计检测荧光极化值,分析荧光极化值随蛋白或多肽浓度变化而改变的趋势。结果:对应于Bak蛋白BH3结构域的5FAM标记肽(LP)可与Bcl-2蛋白相互作用,建立了二者相互作用的饱和曲线;非标记竞争性短肽(CP)和LP竞争性地与Bcl-2蛋白结合,而无关肽(NP)则不与Bcl-2相互作用;基于Bcl-2结构筛选到的小分子化合物SC对体系内LP荧光极化值的影响模式与CP相同。结论:在均相系统内初步建立了基于抑制Bcl-2活性进而促进细胞凋亡的药物高通量筛选方法,为实现药物的高通量筛选奠定了基础。Objective： To construct the high throughput screening method based on fluorescence polarization and the pattern of interaction between Bcl-2 and Bak in liquid system. Methods： Fluorescence polarization signals were detected from solutions in the microtiter-plate format, which is suitable for high throughput screening. The relation between the values of fluorescence polarization and solution of protein or peptide was analyzed. Results： The purified Bcl-2 could interact with the 5FAM-labeled peptide （LP） corresponding to the SH3 domain of Bak protein. The non-labeled specific peptide （competitive peptide, CP）, but not the scrambled control peptide （unrelated peptide,NP）, could compete with LP causing decrease in fluorescence polarization values. The small molecular compound SC, which was screened based on the structure of Bcl-2, could compete with LP, with the same manner as CP, so the compound SC might interact with Bcl-2 at the same site as BH3 peptide. Conclusion： This work might provide the basis for high throughput screening of anti-tumor drugs based on the structure and activity inhibition of Bcl-2.

关 键 词：荧光极化 BCL-2蛋白 BAK蛋白 BH3结构域 饱和曲线 

分 类 号：R965.1[医药卫生—药理学]