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

Authors: Mao-rong ZHU1,2, Dao-hai DU2, Jun-chi HU2, Lian-chun LI2, Jing-qiu LIU2, Hong DING2, Xiang-qian KONG2, Hua-liang JIANG2, Kai-xian CHEN2, Cheng LUO2,3
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 3CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Corresponding to: Xiang-qian KONG:, Cheng LUO:,
DOI: 10.1038/aps.2017.59
Received: 19 February 2017
Accepted: 20 April 2017
Advance online: 31 August 2017


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.
Keywords: PRC2; EZH2; EED; protein-protein interaction; fluorescence polarization; high-throughput screening; apomorphine hydrochloride; oxyphenbutazone; nifedipine; ergonovine maleate