Home Epigenetics Histone Methyltransferase inhibitor - DOT1 inhibitor - Apoptosis related activator EPZ004777

EPZ004777

Catalog No.S7353

For research use only.

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.

EPZ004777 Chemical Structure

CAS No. 1338466-77-5

Selleck's EPZ004777 has been cited by 24 publications

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Biological Activity

Description 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.
Targets
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, EPZ004777 selectively inhibits proliferation of MLL-Rearranged cell lines and MLL-AF9-transformed murine hematopoietic cells. In addition, EPZ004777 also induces differentiation and apoptosis in MLL-rearranged cells. [1] EPZ004777 selectively inhibits proliferation of MLL–AF10 and CALM–AF10-transformed murine bone marrow cells. [2] DOT1L inhibition by EPZ004777 results in significantly decreased proliferation, decreased expression of MLL-AF6 target genes, and cell cycle arrest of MLL-AF6-transformed cells. [3]

Cell Data
Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
MOLM13 MlXaRY51cXC{b3zp[oVz[XSrdnWgZZN{[Xl? NV3CSIp{SW62aYDyc4xq\mW{YYTpeoUh[WO2aY\peJkh[WejaX7zeEBpfW2jbjDNU2xOOTNiY3XscJMh[2:wdHHpcolv\yCPTFytRWY6NCCHQ{WwQVAvODB2zszNMi=> MkLZQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjN6N{m0OlMoRjJ|OEe5OFY{RC:jPh?=
MV4-11 MYPBcpRqeHKxbHnm[ZJifGm4ZTDhd5NigQ>? NGrNXXBCdnSrcILvcIln\XKjdHn2[UBi[3Srdnn0fUBi\2GrboP0JIh2dWGwIF3WOE0yOSClZXzsd{Bkd262YXnubY5oKE2OTD3BSlQtKEWFNUC9NE4xODUQvF2u M2XBelxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ|OEe5OFY{Lz5{M{i3PVQ3OzxxYU6=
THP1 MXPBcpRqeHKxbHnm[ZJifGm4ZTDhd5NigQ>? M1fVc2FvfGmycn;sbYZmemG2aY\lJIFkfGm4aYT5JIFo[Wmwc4SgbJVu[W5iVFjQNUBk\WyuczDjc451[WmwaX7nJG1NVC2DRkmsJGVEPTB;MD6wNFTPxE1w M2jFfFxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ|OEe5OFY{Lz5{M{i3PVQ3OzxxYU6=
MCF10A M1jnRmZ2dmO2aX;uJIF{e2G7 Ml;YTY5pcWKrdHnvckBw\iCGT2SxUEBqdiCqdX3hckBOS0ZzMFGgZ4VtdHNiYYPz[ZN{\WRiYYOgdoVlfWO2aX;uJI9nKEh|S{e5JIxmfmWuLDDJR|UxRTBwMEi0{txONg>? M2jadVxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ3NEC2PFU{Lz5{NUSwOlg2OzxxYU6=
MLL MYnGeY5kfGmxbjDhd5NigQ>? NYHIXFN5UW6qaXLpeIlwdiCxZjDN[Yl{OSCpZX7lJIV5eHKnc4Ppc44hcW5iaIXtZY4hVUyOIHPlcIx{NCCHQ{WwQVAvP87:TT6= MWe8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zOzh5OUS2N{c,OjN6N{m0OlM9N2F-
MLL MnHDSpVv[3Srb36gZZN{[Xl? NYiwWJVxUW6qaXLpeIlwdiCxZjDIc5hiQSCpZX7lJIV5eHKnc4Ppc44hcW5iaIXtZY4hVUyOIHPlcIx{NCCHQ{WwQVAvP87:TT6= MnfQQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOjN6N{m0OlMoRjJ|OEe5OFY{RC:jPh?=
Sf9 NUWzcGFwTnWwY4Tpc44h[XO|YYm= MmHGTY5pcWKrdHnvckBw\iCqdX3hckBnfWyuIHzlcod1cCCSUl3UO{BmgHC{ZYPz[YQhcW5iU3[5JINmdGy|LDDJR|UxRTdwNd88UU4> M3\6NlxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ3OEmzNFQyLz5{NUi5N|A1OTxxYU6=
BHK NFLhNXVCdnSrdnnyZYwh[XO|YYm= M1;CTVgh\GG7cx?= MXzBcpRqfmm{YXygZYN1cX[rdImgZYdicW6|dDDabYtiKH[rcoXzJHNOT0NiaX7m[YN1\WRiaX6gRmhMKGOnbHzzJIF{e2W|c3XkJIF{KGmwaHnibZRqd25ib3[geolzfXNiaX7keYNm\CCleYTvdIF1cGmlIHXm[oVkfCCjZoTldkA5KGSjeYOgZpkhS2WubGTpeIVzNUeubzDseY1qdmW|Y3XueEBk\WyuII\pZYJqdGm2eTDhd5NigSxiSVO1NF0{PS5zOd88UU4> M{XVZVxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzNyMUewN|IyLz5|MEG3NFMzOTxxYU6=
MV4-11 M2PsUGZ2dmO2aX;uJIF{e2G7 NUHMPFdDPiCmYYnz MoHTTY5pcWKrdHnvckBw\iCGT2SxUEBqdiCqdX3hckBOXjRvMUGgZ4VtdHNiYYPz[ZN{\WRiYYOg[I94dnKnZ4XsZZRqd25ib3[gTG9ZSTlxTVXJV|EhdVKQQTDlfJBz\XO|aX;uJIFnfGW{IE[g[IF6eyCkeTDy[YFtNXSrbXWgVGNTKGGwYXz5d4l{NCCLQ{WwQVAvP87:TT6= NHruPWc9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;3e5cv\WKrLnHjMpVsN2OqZX3icE9kd22yb4Xu[H9z\XCxcoTfZ4Fz\C:FSFXNRmwzOTZ7OUG5M{c,S2iHTVLMQE9iRg>?
MOLM13 NUTHblY6TnWwY4Tpc44h[XO|YYm= M2C4bVYh\GG7cx?= NHThZ5ZKdmirYnn0bY9vKG:oIFTPWFFNKGmwIHj1cYFvKE2RTF2xN{Bk\WyuczDhd5Nme3OnZDDhd{Bld3ewcnXneYxifGmxbjDv[kBJV1iDOT;NSWlUOSCvUl7BJIV5eHKnc4Ppc44h[W[2ZYKgOkBl[Xm|IHL5JJJm[WxvdHnt[UBRS1JiYX7hcJl{cXNuIFnDOVA:OC55zszNMi=> NXXmNXRrRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:ve5d4NmWkaT7hZ{52cy:laHXtZoww[2:vcH;1coRgemWyb4L0Y4NiemRxQ1jFUWJNOjF4OUmxPU8oRkOqRV3CUFww[T5?
Click to View More Cell Line Experimental Data
Assay
Methods Test Index PMID
Western blot H3K79me2 ; H3K79me1 / H3K4me3 / H3K9me3 / H3R17me2a / H3K27me2 / H3K27me3 / H3K36me2 / H4R3me2s / H4K20me2 23138183 21741596
Growth inhibition assay Cell viability 25596271
In vivo EPZ004777 produces potent antitumor efficacy, and significantly increases median survival in a mouse xenograft model of MLL leukemia. [1]

Protocol (from reference)

Kinase Assay:

[1]
  • 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 final 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 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.
Cell Research:

[1]
  • Cell lines: Human leukemia cell lines MV4-11, THP-1, RS4;11, Kasumi-1, HL-60, REH, and Jurkat. SEM, KOPN-8, 697, U937 and MOLM-13 cells.
  • Concentrations: ~50 μM
  • Incubation Time: 14-18 days
  • Method:

    For assessment of cell proliferation and viability in human cell lines, exponentially growing cells are plated, in triplicate, in 96-well plates in a final volume of 150 ml. 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 according to the manufacturer’s protocol. 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 continues until IC50 values stabilized (day 18 for THP-1 cells, day 14 for all other cell lines). For assessment of the effect of EPZ004777 treatment on transformed murine hematopoietic progenitors, cells from two independent transductions for each virus are plated in 24-well plates at a density of 0.5–1×105 cell/well in 1 ml media in 24-well plates and exposed to increasing concentrations of EPZ004777 up to 30 mM. Cells are counted and replated at equal cell numbers in fresh media with fresh compound every 3–4 days. For MTT assays, cells from serial replatings are harvested on day 10 and plated, in triplicate at 2×104 cells/well in 100 ml media with the appropriate concentration of EPZ004777. Cells are incubated for 2.5 days, the exposed to 10 ml MTT reagent for 3 hr, and lysed over night in 100 ml MTT- solubilization buffer (both from Cell Proliferation Kit I [MTT]).
Animal Research:

[1]
  • Animal Models: A mouse xenograft MV4-11 model of MLL.
  • Dosages: Mini-pumps containing 100 and 150 mg/mL EPZ004777 solutions
  • Administration: Osmotic pump

Solubility (25°C)

In vitro

Chemical Information

Molecular Weight 539.67
Formula

C28H41N7O4
CAS No. 1338466-77-5
Storage 3 years -20°C powder
2 years -80°C in solvent
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

In vivo Formulation Calculator (Clear solution)

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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.

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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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