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.