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EPZ004777 DOT1L Inhibitor

Name: EPZ004777
SKU: S7353
Availability: InStock
Rating: 5 (32 reviews)

Cat.No.S7353

EPZ004777 is a potent, selective DOT1L inhibitor with IC50 of 0.4 nM in a cell-free assay and demonstrates >1,200-fold selectivity for DOT1L over all other tested PMTs. EPZ004777 induces apoptosis.

Chemical Structure

Molecular Weight: 539.67

Jump to Mechanism of Action Cell Culture & Working Concentration Solubility Chemical Information, Storage & Stability Specificity

Quality Control

Batch: Purity: 99.09%

99.09

Related Targets	Epigenetic Reader Domain HDAC JAK AMPK PKC PARP HIF Histone Acetyltransferase Aurora Kinase Sirtuin PRMT Histone Demethylase EZH1/2 DNA Methyltransferase Pim LSD1 MLL JMJD G9a/GLP NSD FTO MicroRNA PAD SETD SMYD DOT1 COMT WDR5 HNMT SAM MTase
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Cell Culture, Treatment & Working Concentration

Cell Lines	Assay Type	Incubation Time	Activity Description	PMID
MOLM13	Antiproliferative assay		Antiproliferative activity against human MOLM13 cells containing MLL-AF9, EC50=0.004μM.	23879463
MV4-11	Antiproliferative assay		Antiproliferative activity against human MV4-11 cells containing MLL-AF4, EC50=0.004μM.	23879463
THP1	Antiproliferative assay		Antiproliferative activity against human THP1 cells containing MLL-AF9, EC50=0.004μM.	23879463
MCF10A	Function assay		Inhibition of DOT1L in human MCF10A cells assessed as reduction of H3K79 level, IC50=0.084μM.	25406853
MLL	Function assay		Inhibition of Meis1 gene expression in human MLL cells, EC50=0.7μM.	23879463
MLL	Function assay		Inhibition of Hoxa9 gene expression in human MLL cells, EC50=0.7μM.	23879463
Sf9	Function assay		Inhibition of human full length PRMT7 expressed in Sf9 cells, IC50=7.5μM.	25893041
BHK	Antiviral assay	8 days	Antiviral activity against Zika virus SMGC infected in BHK cells assessed as inhibition of virus induced cytopathic effect after 8 days by CellTiter-Glo luminescent cell viability assay, IC50=35.19μM.	30170321
MV4-11	Function assay	6 days	Inhibition of DOT1L in human MV4-11 cells assessed as downregulation of HOXA9/MEIS1 mRNA expression after 6 days by real-time PCR analysis, IC50=0.7μM.	ChEMBL
MOLM13	Function assay	6 days	Inhibition of DOT1L in human MOLM13 cells assessed as downregulation of HOXA9/MEIS1 mRNA expression after 6 days by real-time PCR analysis, IC50=0.7μM.	ChEMBL
Click to View More Cell Line Experimental Data

Chemical Information, Storage & Stability

Molecular Weight	539.67	Formula	C₂₈H₄₁N₇O₄	Storage (From the date of receipt)	3 years-20°Cpowder
CAS No.	1338466-77-5	Download SDF		Storage of Stock Solutions	1 year-80°Cin solvent 1 month-20°Cin solvent
Synonyms	N/A			Smiles	CC(C)N(CCCNC(=O)NC1=CC=C(C=C1)C(C)(C)C)CC2C(C(C(O2)N3C=CC4=C(N=CN=C43)N)O)O

Solubility

In vitro
Batch:

DMSO : 100 mg/mL (185.29 mM)
(Moisture-contaminated DMSO may reduce solubility. Use fresh, anhydrous DMSO.)

Ethanol : 100 mg/mL

Water : Insoluble

Molarity Calculator

Mass	Concentration	Volume	Molecular Weight
=	×	×

Dilution Calculator Molecular Weight Calculator

In vivo Batch:
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Clear solution	5%DMSO 1 Corn oil Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	5.000mg/ml (9.26mM)	Taking the 1 mL working solution as an example, add 50 μL of 100 mg/ml clear DMSO stock solution to 950 μL of corn oil and mix evenly. The mixed solution should be used immediately for optimal results.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Clear solution	5%DMSO 1 40%PEG300 2 5%Tween80 3 50%ddH2O Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	5.000mg/ml (9.26mM)	Taking the 1 mL working solution as an example, add 50 μL of 100 mg/ml clarified DMSO stock solution to 400 μL of PEG300, mix evenly to clarify it; add 50 μL of Tween80 to the above system, mix evenly to make it clear; then continue to add 500 μL of ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)

Dosage: mg/kg Average weight of animals: g Dosing volume per animal:μL Number of animals:

Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)

% DMSO + % + % Tween 80 + % ddH₂O

%DMSO+ %

Calculation results:

Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such
as vortex, ultrasound or hot water bath can be used to aid dissolving.

Mechanism of Action

Targets/IC₅₀/Ki	DOT1L ^[1] (Cell-free assay) 0.4 nM
In vitro	EPZ004777 selectively inhibits cellular H3K79 methylation and inhibits expression of key MLL fusion target genes. Following DOT1L inhibition, this compound selectively inhibits proliferation of MLL-Rearranged cell lines and MLL-AF9-transformed murine hematopoietic cells. In addition, it also induces differentiation and apoptosis in MLL-rearranged cells. ^[1] This chemical selectively inhibits proliferation of MLL–AF10 and CALM–AF10-transformed murine bone marrow cells. ^[2] DOT1L inhibition by this agent results in significantly decreased proliferation, decreased expression of MLL-AF6 target genes, and cell cycle arrest of MLL-AF6-transformed cells. ^[3]
Kinase Assay	Determination of Inhibitor IC50 Values
Kinase Assay	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 ﬁnal concentration of the product inhibitor S-adenosyl-L-homocysteine, (SAH). This 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 ﬁnal 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 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.
In vivo	EPZ004777 produces potent antitumor efficacy, and significantly increases median survival in a mouse xenograft model of MLL leukemia. ^[1]
References	[1] https://pubmed.ncbi.nlm.nih.gov/21741596/ [2] https://pubmed.ncbi.nlm.nih.gov/23138183/ [3] https://pubmed.ncbi.nlm.nih.gov/23361907/

Applications

Methods	Biomarkers	Images	PMID
Western blot	H3K79me2 H3K79me1 / H3K4me3 / H3K9me3 / H3R17me2a / H3K27me2 / H3K27me3 / H3K36me2 / H4R3me2s / H4K20me2		23138183
Growth inhibition assay	Cell viability		25596271

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