A Fusion Protein of the p53 Transaction Domain and the p53-Binding Domain of the Oncoprotein MdmX as an Efficient System for High-Throughput Screening of MdmX Inhibitors
A Fusion Protein of the p53 Transaction Domain and the p53-Binding Domain of the Oncoprotein MdmX as an Efficient System for High-Throughput Screening of MdmX Inhibitors
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p53 交易结构域和癌蛋白 MdmX 的 p53 结合结构域的融合蛋白作为高通量筛选 MdmX 抑制剂的有效系统
DOI：
10.1021/acs.biochem.7b00085
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发表时间：
2017-06-27
期刊：
影响因子：
2.9
通讯作者：
Su, Zhengding
中科院分区：
文献类型：
--
作者：
Chen, Rong;Zhou, Jingjing;Su, Zhengding
在近一半的癌症中，p53蛋白的抗癌活性经常被过表达的癌蛋白Mdm 2及其同源物MdmX损害，需要有效的治疗方法来破坏异常的p53-MdmX/Mdm 2相互作用以恢复p53活性。虽然许多有效的Mdm 2特异性抑制剂已经进行了临床研究，但寻找MdmX特异性抑制剂已经变得非常有吸引力，需要更有效的筛选策略来评估潜在的支架或先导物。在这项工作中，考虑到内在荧光残基Trp 23在决定p53-MdmX/Mdm 2相互作用中起着重要作用，我们构建了一个融合蛋白，利用这个内在荧光信号来监测化合物库的高通量筛选。融合蛋白由p53 p和MdmX的N-末端结构域（N-MdmX）通过柔性氨基酸接头组成，而整个融合蛋白含有唯一的内源荧光探针。然后使用荧光光谱法针对模型化合物评估融合蛋白。我们的结果显示，荧光信号的变化与65内的配体的浓度高度相关。融合蛋白进一步评估其用于使用模型化合物文库的高通量筛选的可行性;包括对照。我们发现，咪唑并吲哚支架是一个真正的基于模板的MdmX抑制剂的设计支架。因此，我们设计的p53 p N-MdmX融合蛋白为高通量筛选新的MdmX抑制剂提供了一个方便和有效的工具。这项工作中描述的策略应该适用于其他蛋白质靶点，以加速药物发现。
In nearly half of cancers,. the anticancer activity of p53 protein is often impaired by the overexpressed oncoprotein Mdm2 and its homologue, MdmX, demanding efficient therapeutics to disrupt the aberrant p53-MdmX/Mdm2 interactions to restore the p53 activity. While many :potent Mdm2-specific inhibitors have already :undergone clinical investigations, searching for MdmX-specific inhibitors has become very attractive, requiring a more efficient screening strategy for evaluating potential scaffolds or leads. In this work, considering that the intrinsic fluorescence residue Trp23 in the p53 transaction domain (p53p) plays an important role in determining the p53-MdmX/Mdm2 interactions, we constructed a fusion protein to utilize this intrinsic fluorescence signal to monitor high-throughput screening of a compound library. The fusion protein was composed of the p53p followed, by the N-terminal domain of MdmX (N-MdmX) through a flexible amino acid linker, while the whole fusion protein contained a sole intrinsic fluorescence probe. The fusion protein was then evaluated using fluorescence spectroscopy against model compounds. Our results revealed that the variation of the fluorescence signal was highly correlated with the concentration of the ligand within 65 The fusion protein was further evaluated with respect to its feasibility for use in high-throughput screening using a model compound library; including controls. We found that the imidazo-indole scaffold was a bona fide scaffold for template-based design of MdmX inhibitors. Thus, the p53p N-MdmX fusion protein we designed provides a convenient and efficient tool for high-throughput screening of new MdmX inhibitors. The strategy described in this work should be applicable for other protein targets to accelerate drug discovery.