EPZ004777 HCl

Determination of Inhibitor IC50 Values

EPZ004777 is serially diluted 3-fold in DMSO for a total of ten concentrations, beginning at 1 μM. A 1 μL aliquot of each inhibitor dilution is plated in a 384-well microtiter plate. The 100% inhibition control consisted of 2.5 μM final concentration of the product inhibitor S-adenosyl-L-homocysteine, (SAH). Compound is incubated for 30 min with 40 μl 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 final reaction mixture at their respective KM values). Reactions are incubated for 120 min and quenched with 10 μl per well of 800 μM SAM. Incorporation of radioactivity into nucleosome substrate is measured in a flashplate. 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.

Cell lines

MV4-11 cells, MOLM-13 cells, MLL–AF10 and CALM–AF10 transformed bone marrow cells

Preparation method

This compound is soluble in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

3, 10, 50 μM for 6-18 days;

Applications

EPZ004777 HCl selectively inhibited cellular H3K79 methylation, blocked leukemogenic gene expression, and selectively killed Mixed lineage leukemia (MLL)-rearranged leukemic cells (MV4-11 and MOLM-13 cells) [1]. Moreover, EPZ004777 HCl suppressed the expression of leukemogenic genes including Hoxa cluster genes and Meis1, and selectively inhibited MLL–AF10 and CALM–AF10 transformed cells proliferation [2].

Animal models

Mouse Mixed lineage leukemia (MLL) xenograft model

Dosage form

50, 100, or 150 mg/ml; osmotic pump for 14 days

Applications

EPZ004777 HCl administration showed antitumor activity and induced the extension of survival in a mouse MLL xenograft model [1]. Moreover, EPZ004777 HCl effectively decreased the spleen-colony-forming ability of MLL–AF10 or CALM–AF10 transformed bone marrow cells in vivo [2].

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

1Daigle, S. R., Olhava, E. J., Therkelsen, C. A., Majer, C. R., Sneeringer, C. J., Song, J., Johnston, L. D., Scott, M. P., Smith, J. J., Xiao, Y., Jin, L., Kuntz, K. W., Chesworth, R., Moyer, M. P., Bernt, K. M., Tseng, J. C., Kung, A. L., Armstrong, S. A., Copeland, R. A., Richon, V. M. and Pollock, R. M. (2011) Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell. 20, 53-65

2Chen, L., Deshpande, A. J., Banka, D., Bernt, K. M., Dias, S., Buske, C., Olhava, E. J., Daigle, S. R., Richon, V. M., Pollock, R. M. and Armstrong, S. A. (2013) Abrogation of MLL-AF10 and CALM-AF10-mediated transformation through genetic inactivation or pharmacological inhibition of the H3K79 methyltransferase Dot1l. Leukemia. 27, 813-822