EPZ005687 

10-point curves of EPZ005687 are made using serial 3-fold dilution in DMSO, beginning at 2.5 mM (final top concentration of compound is 50 μM and the DMSO is 2%). A 1 μL aliquot the inhibitor dilution series is spotted in a 384-well microtiter plate. The 100% inhibition control consisted of 1 mM final concentration of the product inhibitor S-adenosylhomocysteine (SAH). Compound is incubated for 30 min with 40 μL per well of 5 nM PRC2 (final assay concentration in 50 μL is 4 nM) in 1X assay buffer (20 mM Bicine [pH 7.6], 0.002% Tween 20, 0.005% Bovine Skin Gelatin and 0.5 mM DTT). 10 μL per well of substrate mix comprising assay buffer ³H-SAM, unlabeled SAM, and peptide representing histone H3 residues 21-44 containing C-terminal biotin (appended to a C-terminal amide-capped lysine) are added to initiate the reaction. Reactions are incubated for 90 min at room temperature and quenched with 10 μL per well of 600 μM unlabeled SAM, then transferred to 384-well Flashplate and washed after 30 min.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

For cell cycle, WSU-DLCL2 cells are plated in 12-well plates at a density of 1×10⁵ cells per mL. Cells are incubated with EPZ005687 at 0.2 μM, 0.67 μM, 2 μM and 6 μM, in a total of 2 mL, over a course of 10 d. All remaining cell cycle analysis is performed.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Knutson SK, et al. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat Chem Biol. 2012;8(11):890-6.  [Content Brief]

  [2]. Tang SH, et al. Effects of H3K27 methylation inhibitor EPZ005687 on apoptosis, proliferation and cell cycle of U937 cells and normal CD34 positive cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2014 Dec;22(6):1561-6.  [Content Brief]