1. Epigenetics
  2. Histone Methyltransferase

EPZ004777 

Cat. No.: HY-15227 Purity: 99.89%
Data Sheet SDS Handling Instructions

EPZ004777 is a potent, selective DOT1L inhibitor with IC50 of 0.4 nM.

For research use only. We do not sell to patients.
EPZ004777 Chemical Structure

EPZ004777 Chemical Structure

CAS No. : 1338466-77-5

Size Price Stock Quantity
Free Sample (0.5-1 mg)   Apply now  
10 mM * 1 mL in DMSO $131 In-stock
5 mg $110 In-stock
10 mg $190 In-stock
50 mg $550 In-stock
100 mg $940 In-stock
200 mg   Get quote  
500 mg   Get quote  

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Customer Review

Other Forms of EPZ004777:

  • Biological Activity

  • Protocol

  • Technical Information

  • Purity & Documentation

  • References

Description

EPZ004777 is a potent, selective DOT1L inhibitor with IC50 of 0.4 nM.

IC50 & Target

IC50: 0.4 nM (DOT1L)[1]

In Vitro

EPZ004777 demonstrates potent, concentration-dependent inhibition of DOT1L enzyme activity with an IC50 of 400±100 pM. EPZ004777 displays remarkable selectivity for inhibition of DOT1L over other HMTs(PRMT5, 521±137 nM; others, >50 μM). The effect of extended EPZ004777 treatment is remarkably specific for the MLL-rearranged cell lines. The number of viable MV4-11 and MOLM-13 cells is dramatically reduced by EPZ004777, whereas the growth of Jurkat cells is unaffected. A small population of MV4-11 cells remain viable in the presence of EPZ004777, but their number remain constant when growth curves are tracked over longer periods indicating that they have ceased to divide. The proliferation of MLL-AF9-transformed cells is strongly inhibited by EPZ004777 at concentrations of 3 μM or greater[1]. EPZ004777 selectively inhibits proliferation of MLL-AF10 and CALM-AF10 transformed murine bone marrow cells[2].

In Vivo

EPZ004777 is well tolerated and no overt toxicity is observed. Complete blood count analysis after 14 days of continuous exposure to EPZ004777 revealed a statistically significant increase in the total white blood cell count, which resulted from an increase in neutrophils, monocytes, and lymphocytes. EPZ004777 (50, 100, or 150 mg/mL) administration is well tolerated, and no significant weight loss is observed[1].

References
Preparing Stock Solutions
Concentration Volume Mass 1 mg 5 mg 10 mg
1 mM 1.8530 mL 9.2649 mL 18.5298 mL
5 mM 0.3706 mL 1.8530 mL 3.7060 mL
10 mM 0.1853 mL 0.9265 mL 1.8530 mL
Please refer to the solubility information to select the appropriate solvent.
Kinase Assay
[1]

Avian (chicken) erythrocyte oligonucleosomes are purified. 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 μL per well of substrate mix comprising assay buffer with 200 nM 3H-SAM (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, an assay format referred to as “balanced conditions”. 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[1]. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Cell Assay
[1]

EPZ004777 is prepared in DMSO (50 or 10 mM) and stored (−20°C), and then diluted with appropriate medium (DMSO 0.2%) before use[1].

For assessment of cell proliferation and viability in human cell lines, exponentially growing cells are plated, in triplicate, in 96-well plates at a density of 3×104 cells/well in a final volume of 150 μL. Cells are incubated in the presence of 3 μM (proliferation curve), or increasing concentrations (IC50 determination) of EPZ004777 up to 50 μM. Viable cell number is determined every 3-4 days for up to 18 days using the Guava Viacount assay and analyzed on a Guava EasyCyte Plus instrument. On days of cell counts, growth media and EPZ004777 are replaced and cells split back to a density of 5×104 cells/well. Total cell number is expressed as split-adjusted viable cells per well. For each cell line, IC50 values are determined from concentration-dependence curves at each time point using Graphpad Prism software. Experiments to determine IC50 values continued until IC50 values stabilized (day 18 for THP-1 cells, day 14 for all other cell lines)[1]. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Administration
[1]

EPZ004777 is prepared in 10% ethanol and 90% water (Mice)[1].

Mice[1]
Nine-week-old female nude mice (nu/nu) are injected subcutaneously with MV4-11 cells in the right flank (200 μL of a 5×107 cells/mL suspension in a 1:1 mixture of PBS and Matrigel). Mice are randomized to treatment groups when tumor sizes reached 300-400 mm3. Six mice received subcutaneous implant of osmotic pumps, containing 50 mg/mL EPZ004777 in 10% ethanol, 90% water, and five control mice received no pump implant. Six days after pump implant, animals are sacrificed and tumor samples from treated and control animals are collected for immunoblot analysis. For the disseminated leukemia model, MV4-11 cells are transduced with the pMMP-LucNeo retrovirus. Eight-week-old female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice are purchased from Jackson Laboratories. A total of 1×107 MV4-11-LucNeo cells are injected intravenously via the lateral tail vein. Engraftment of disseminate leukemia is determined by bioluminescence imaging after injection of 75 mg/kg of D-luciferin. Animals with documented leukemia are divided into treatment groups consisting of vehicle (15% ethanol, 50% PEG300, 35% water) loaded osmotic pumps, or EPZ004777 at 50, 100, or 150 mg/mL. Osmotic pumps are replaced after one week. Irritation caused by compound precipitation is observed in the 100 and 150 mg/mL dose groups, precluding additional pump replacements. Animals are monitored daily for clinical symptoms, and are euthanized when they displayed signs of distress consistent with terminal leukemic disease. Log-rank analysis is used to determine statistical significance of the survival curves. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

References
Molecular Weight

539.67

Formula

C₂₈H₄₁N₇O₄

CAS No.

1338466-77-5

Storage
Powder -20°C 3 years
  4°C 2 years
In solvent -80°C 6 months
  -20°C 1 month
Shipping

Room temperature in continental US; may vary elsewhere

Solvent & Solubility

10 mM in DMSO

* "<1 mg/mL" means slightly soluble or insoluble. "≥" means soluble, but saturation unknown.

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Product Name:
EPZ004777
Cat. No.:
HY-15227
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