B1581-5mg | EPZ004777 HCl [1380316-03-9] Clinisciences

Background

EPZ004777 is a potent and selective inhibitor of DOT1L, a protein methyltransferase catalyzing the methylation of lysine 4 of histone H3 (H3K4), with the half maximal inhibition concentration IC50 value of 400 pM [1].

EPZ004777 has been found to concentration-dependently inhibit the H3K79 methylation in a variety of mixed lineage leukemia (MLL) cells, including MLL-rearranged acute myeloid leukemia (MOLM-13 and MLL-AF9), MLL-rearranged biphenotypic leukemia (MV4-11 and MLL-AF4) and non-MLL-rearranged T cell acute leukemia cells [1].

Additionally, EPZ004777 exhibits anti-proliferative activity against both MLL-rearranged and non-rearranged human leukemia cell lines, including RS4;11 (IC50: 6.47 μM), SEM (IC50: 1.72 μM), MV4-11 (IC50: 0.17 μM), THP-1 (IC50: 3.36 μM), MOLM-13 (IC50: 0.72 μM), KOPN-8 (IC50: 0.62 μM), REH (IC50: 13.9 μM), Kasumi-1 (IC50: 32.99 μM) and 697 (IC50: 36.57 μM) [1].

References:
[1] Daigle SR, Olhava EJ, Therkelsen CA, Majer CR, Sneeringer CJ, Song J, Johnston LD, Scott MP, Smith JJ, Xiao Y, Jin L, Kuntz KW, Chesworth R, Moyer MP, Bernt KM, Tseng JC, Kung AL, Armstrong SA, Copeland RA, Richon VM, Pollock RM. Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell. 2011 Jul 12;20(1):53-65. doi: 10.1016/j.ccr.2011.06.009.

Chemical Properties

Storage	Store at -20°C
M.Wt	576.13
Cas No.	1380316-03-9
Formula	C₂₈H₄₁N₇O₄·HCl
Solubility	insoluble in H2O; ≥16.5 mg/mL in DMSO; ≥26.35 mg/mL in EtOH
Chemical Name	1-[3-[[(2R,3S,4R,5R)-5-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxyoxolan-2-yl]methyl-propan-2-ylamino]propyl]-3-(4-tert-butylphenyl)urea;hydrochloride
SDF	Download SDF
Canonical SMILES	CC(C)N(CCCNC(=O)NC1=CC=C(C=C1)C(C)(C)C)CC2C(C(C(O2)N3C=CC4=C3N=CN=C4N)O)O.Cl
Shipping Condition	Small Molecules with Blue Ice, Modified Nucleotides with Dry Ice.
General tips	We do not recommend long-term storage for the solution, please use it up soon.

Protocol

Kinase experiment [1]:
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 experiment:
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 experiment:
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

Biological Activity

Description	EPZ004777 HCl is a potent, selective inhibitor of DOT1L with an IC50 value of 0.4 nM.
Targets	DOT1L
IC50	0.4 nM

Quality Control

Purity = 99.83%

Chemical structure

EPZ004777 HCl [1380316-03-9]

Referentie B1581-5mg

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