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

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Bd-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。

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

Objective:To construct the h igh throughput screen ing m ethod based on fluorescence polarization and the pattern of interaction between Bc l-2 and Bak in liqu id system.M ethod s:F luorescence polarization signals were detected from solutions in the m icrotiter-p late form at,wh ich is su itab le for h igh throughput screen ing.The relation between the values of fluorescence polarization and solution of prote in or peptide was analyzed.R esu lts:The purified Bc l-2 cou ld interactw ith the 5FAM-labeled peptide(LP) correspond ing to the SH3 dom ain of Bak prote in.The non-labeled spec ific peptide(com-petitive peptide,CP),but not the scramb led control peptide(unrelated peptide,NP),cou ld compete w ith LP causing de-crease in fluorescence polarization values.The sm allmolecu lar compound SC,wh ich was screened based on the structure of Bc l-2,cou ld compete w ith LP,w ith the sam e m anner as CP,so the compound SC m ight interactw ith Bc l-2 at the sam e site as BH3 peptide.C onc lu sion:Th is work m ight provide the basis for h igh throughput screen ing of anti-tumor drugs based on the structure and activity inh ib ition of Bc l-2.