Screening of Small-Molecule Inhibitors of Protein-Protein Interaction with Capillary Electrophoresis Frontal Analysis
Screening of Small-Molecule Inhibitors of Protein-Protein Interaction with Capillary Electrophoresis Frontal Analysis
复制标题
毛细管电泳前沿分析筛选蛋白质-蛋白质相互作用的小分子抑制剂
DOI：
10.1021/acs.analchem.6b01430
复制
发表时间：
2016
影响因子：
7.4
通讯作者：
Kang Jingwu
中科院分区：
文献类型：
--
作者：
Xu Mei;Liu Chao;Zhou Mi;Li Qing;Wang Renxiao;Kang Jingwu
利用毛细管电泳前沿分析技术（CE-FA）建立了一种简单有效的蛋白质相互作用抑制剂（PPI）的鉴定方法。抗凋亡B细胞-2（Bcl-2）家族成员Bcl-XL蛋白、作为配体的从Bid的BH 3结构域截短的5-羧基荧光素标记的肽（F-Bid）和已知的Bcl-XL-Bid相互作用抑制剂ABT-263被用作用于概念证明的实验模型。在CE-FA中，将游离配体与蛋白质和蛋白质-配体复合物分离，以允许测量配体的平衡浓度，从而测量蛋白质-配体复合物的解离常数。在抑制剂的存在下，蛋白质-配体复合物的形成受阻，从而可以通过配体的升高的平台高度和复合物的衰减的平台来容易地识别抑制。此外，我们提出了一个公式，用于转换的IC 50值到抑制常数Ki值，这是比前者更有用的比较。此外，采用样本合并策略，使筛选通量提高了10倍以上。利用该方法筛选了一个由合成化合物和天然提取物组成的小型化合物库，鉴定出两个天然产物，即去甲基泽拉斯特醛和雷公藤红素，它们是阻断Bcl-XL-Bid相互作用的新抑制剂。进行基于细胞的测定以验证所鉴定的化合物的活性。结果表明，CE-FA代表了一种简单而稳健的PPI抑制剂筛选技术。
A simple and effective method for identifying inhibitors of protein–protein interactions (PPIs) was developed by using capillary electrophoresis frontal analysis (CE-FA). Antiapoptotic B-cell-2 (Bcl-2) family member Bcl-XLprotein, a 5-carboxyfluorescein labeled peptide truncated from the BH3 domain of Bid (F-Bid) as the ligand, and a known Bcl-XL-Bid interaction inhibitor ABT-263 were employed as an experimental model for the proof of concept. In CE-FA, the free ligand is separated from the protein and protein–ligand complex to permit the measurement of the equilibrium concentration of the ligand, hence the dissociation constant of the protein–ligand complex. In the presence of inhibitors, formation of the protein–ligand complex is hindered, thereby the inhibition can be easily identified by the raised plateau height of the ligand and the decayed plateau of the complex. Further, we proposed an equation used to convert the IC50value into the inhibition constantKivalue, which is more useful than the former for comparison. In addition, the sample pooling strategy was employed to improve the screening throughput more than 10 times. A small chemical library composed of synthetic compounds and natural extracts were screened with the method, two natural products, namely, demethylzeylasteral and celastrol, were identified as new inhibitors to block the Bcl-XL-Bid interaction. Cell-based assay was performed to validate the activity of the identified compounds. The result demonstrated that CE-FA represents a straightforward and robust technique for screening of PPI inhibitors.