Development of a high-throughput fluorescence polarization assay for the discovery of EZH2-EED interaction inhibitors  

作　　者：Mao-rong ZHU Dao-hai DU Jun-chi HU Lian-chun LI Jing-qiu LIU Hong DING Xiang-qian KONG Hua-liang JIANG Kai-xian CHEN Cheng LUO 

机构地区：[1]Nano Science and Technology Institute, University of Science and Technology of China, Suzhou 215123, China [2]State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China [3]CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

出　　处：《Acta Pharmacologica Sinica》2018年第2期302-310,共9页中国药理学报（英文版）

基　　金：The authors disclose the following financial support for this research and/or authorship of this article： the Ministry of Sci- ence and Technology of China （2015CB910304 to Hua-liang JIANG）, the National Natural Science Foundation of China （21472208 and 81625022 to Cheng LUO, 21210003, 81230076 and 91313000 to Hua-liang JIANG）, and the Fund of the State Key Laboratory of Toxicology and Medical Countermeasures, Academy of Military Medical Science （TMC201505 to Cheng LUO）. We are extremely grateful to the National Centre for Protein Science Shanghai （Shanghai Science Research Center, Protein Expression and Purification System） for providing instrument support and technical assistance.

摘　　要：Aberrant activity of enhancer of zeste homolog 2 （EZH2） is associated with a wide range of human cancers. The interaction of EZH2 with embryonic ectoderm development （EED） is required for EZH2＇s catalytic activity. Inhibition of the EZH2-EED complex thus represents a novel strategy for interfering with the oncogenic potentials of EZH2 by targeting both its catalytic and non-catalytic functions. To date, there have been no reported high-throughput screening （HTS） assays for inhibitors acting at the EZH2-EED interface In this study, we developed a fluorescence polarization （FP）-based HTS system for the discovery of EZH2-EED interaction inhibitors. The tracer peptide sequences, positions of fluorescein labeling, and a variety of physicochemical conditions were optimized. The high Z＇ factors （〉0.9） at a variety of DMSO concentrations suggested that this system is robust and suitable for HTS. The minimal sequence requirement for the EZH2-EED interaction was determined by using this system. A pilot screening of an in-house compound library containing 1600 FDA-approved drugs identified four compounds （apomorphine hydrochloride, oxyphenbutazone, nifedipine and ergonovine maleate） as potential EZH2-EED interaction inhibitors.Aberrant activity of enhancer of zeste homolog 2 （EZH2） is associated with a wide range of human cancers. The interaction of EZH2 with embryonic ectoderm development （EED） is required for EZH2＇s catalytic activity. Inhibition of the EZH2-EED complex thus represents a novel strategy for interfering with the oncogenic potentials of EZH2 by targeting both its catalytic and non-catalytic functions. To date, there have been no reported high-throughput screening （HTS） assays for inhibitors acting at the EZH2-EED interface In this study, we developed a fluorescence polarization （FP）-based HTS system for the discovery of EZH2-EED interaction inhibitors. The tracer peptide sequences, positions of fluorescein labeling, and a variety of physicochemical conditions were optimized. The high Z＇ factors （〉0.9） at a variety of DMSO concentrations suggested that this system is robust and suitable for HTS. The minimal sequence requirement for the EZH2-EED interaction was determined by using this system. A pilot screening of an in-house compound library containing 1600 FDA-approved drugs identified four compounds （apomorphine hydrochloride, oxyphenbutazone, nifedipine and ergonovine maleate） as potential EZH2-EED interaction inhibitors.

关 键 词：PRC2 EZH2 EED protein-protein interaction fluorescencepolarization high-throughput screening apomorphine hydrochloride oxyphenbutazone NIFEDIPINE ergonovine maleate 

分 类 号：TQ113.247[化学工程—无机化工] TS264.21[轻工技术与工程—发酵工程]