SGC0946 is a highly effective and specific DOT1L methyltransferase inhibitor (IC50: 0.3 nM); selectively kill mixed lineage leukemia cells.

SGC 0946 significantly decreases di-methylation of H3K79 in A431 cells (IC50: 2.6 nM) and MCF10A cells (IC50: 8.8 nM), effectively and selectively killing cells with MLL translocations. Compared to its analogue EPZ004777, SGC 0946 is more potent against DOT1L, making it promising for cancer research. Moreover, SGC 0946 demonstrates over 100-fold higher selectivity for DOT1L than other histone methyltransferases (HMTs).

Determination of Inhibitor IC50 Values: EPZ004777 is serially diluted 3-fold in DMSO for a total of ten concentrations, beginning at 1 mM. A 1 μL aliquot of each inhibitor dilution is plated in a 384-well microtiter plate. The 100% inhibition control consisted of 2.5 mM ?nal concentration of the product inhibitor S-adenosyl-L-homocysteine, (SAH). Compound is incubated for 30 min with 40 ml per well of 0.25 nM DOT1L(1-416) in assay buffer (20 mM TRIS [pH 8.0] 10 mM NaCl, 0.002% Tween 20, 0.005% Bovine Skin Gelatin, 100 mM KCl, and 0.5 mM DTT). 10 ml per well of substrate mix comprising assay buffer with 200 nM 3H-SAM (American Radiolabeled Chemicals: 80 Ci/mmol), 600 nM unlabeled SAM, and 20 nM nucleosomes are added to initiate the reaction (both substrates are present in the ?nal reaction mixture at their respective KM values). Reactions are incubated for 120 min and quenched with 10 ml per well of 800 mM SAM. Incorporation of radioactivity into nucleosome substrate is measured in a ?ashplate. IC50 values for enzymes in the histone methyltransferase panel are determined under similar balanced assay conditions with both SAM and protein/peptide substrate present at concentrations equal to their respective KM values.