Structure, binding, and inhibitory activity of EPZ-5676. (A) Chemical structure of EPZ-5676. (B) Superposition of DOT1L-EPZ-5676 (green) and DOT1L-SAM (gray; Protein Data Base accession number 3QOW). To accommodate the extended hydrophobic tail of the inhibitor, significant rearrangement of the protein is required, including the loop between β-strands 10 and 11 (L10-11). Details on the interactions between the protein and compound and the induced changes caused by EPZ-5676 binding are shown in supplemental Figure 1. (C) Selectivity profile of EPZ-5676 inhibitory activity against representative members of the lysine (left) and arginine (right) enzyme families. The diameter of the sphere for each enzyme is directly related to the magnitude of inhibition by EPZ-5676. Larger circles correlate to increased potency; gray circles indicate no measurable inhibition up to 10 µM of EPZ-5676. (D) Comparison of EPZ-5676 and EPZ004777 potency, selectivity, and cell-based activity. The enzyme inhibition K_i values for EPZ004777 and EPZ-5676 in DOT1L enzymatic assays are listed (n = 3; mean values ± SD are shown). Residence time for each compound is listed and was calculated as the reciprocal of the enzymatic-ligand dissociation rate as determined by surface plasmon resonance. Also listed are inhibitory activities for both compounds in MV4-11 proliferation assays (n = 3 [EPZ-5676] or n = 2 [EPZ004777]; mean values ± SD are shown), MV4-11 cell H3K79me2 ELISA assays (n = 2; mean values ± SD are shown) and MV4-11 cell HOXA9 and MEIS1 qRT-PCR assays (n = 2; mean values ± SD are shown).