EPZ005687, a potent and selective inhibitor of EZH2.

EZH2:24 nM(Ki)

EPZ005687 directly inhibits the enzymatic activity of PRC2 without disrupting the protein-protein interactions among PRC2 subunits. It exhibits concentration-dependent inhibition of PRC2 enzymatic activity with an IC50 value of 54 nM. Additionally, EPZ005687 reduces H3K27 methylation in various lymphoma cell types and demonstrates potent cytotoxic effects in cells harboring Tyr641 or Ala677 mutations, while having minimal impact on the proliferation of wild-type cells. In lymphoma cell lines with the Tyr641 mutation, EPZ005687 causes derepression of known EZH2 target genes and affects genes specifically suppressed by the EZH2 Tyr641 mutant.

Biochemical Enzyme Assays: 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 3 H-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 (both substrates are present in the final reaction mixture at their respective Km values, an assay format referred to as ‘‘balanced conditions''. The final concentrations of substrates and methylation state of the substrate peptide are indicated for each enzyme Reactions are incubated for 90 min at room temperature and quenched with 10 μL per well of 600 μM unlabeled SAM, Then transferred to a 384-well flashplate and washed after 30 min.

Plating densities are determined for each cell line on the basis of linear log-phase growth. Cells are counted and split back to the original plating density in fresh medium with EPZ005687 on days 4 and 7.(Only for Reference)