EPZ004777-hydrochloride - Histone Methyltransferase 抑制劑 - 生命科學試劑 - MedChemExpress

1、Hotline: 400-820-3792Inhibitors Agonists Screening Librarieswww.MedChemEEPZ004777 hydrochlorideCat. No.: HY-15227ACAS No.: 1380316-03-9分式: CHClNO分量: 576.13作靶點: Histone Methyltransferase作通路: Epigenetics儲存式: Please store the product under the recommended conditions inthe COA.BIOLOGICAL ACTIVITY物活性 EPZ

2、004777鹽酸鹽活性DOT1L抑制劑，IC50為0.4 nM。IC50 & Target IC50: 0.4 nM (DOT1L) 1體外研究 EPZ004777 demonstrates potent, concentration-dependent inhibition of DOT1L enzyme activity with an IC50of 400100 pM. EPZ004777 displays remarkable selectivity for inhibition of DOT1L over other HMTs(PRMT5,521137 nM; others, 50

3、M). The effect of extended EPZ004777 treatment is remarkably specific for theMLL-rearranged cell lines. The number of viable MV4-11 and MOLM-13 cells is dramatically reduced byEPZ004777, whereas the growth of Jurkat cells is unaffected. A small population of MV4-11 cells remainviable in the presence

4、 of EPZ004777, but their number remain constant when growth curves are tracked overlonger periods indicating that they have ceased to divide. The proliferation of MLL-AF9-transformed cells isstrongly inhibited by EPZ004777 at concentrations of 3 M or greater 1. EPZ004777 selectively inhibitsprolifer

5、ation of MLL-AF10 and CALM-AF10 transformed murine bone marrow cells 2.體內(nèi)研究 EPZ004777 is well tolerated and no overt toxicity is observed. Complete blood count analysis after 14 daysof continuous exposure to EPZ004777 revealed a statistically significant increase in the total white blood cellcount,

6、which resulted from an increase in neutrophils, monocytes, and lymphocytes. EPZ004777 (50, 100, or150 mg/mL) administration is well tolerated, and no significant weight loss is observed 1.PROTOCOLKinase Assay 1 Avian (chicken) erythrocyte oligonucleosomes are purified. EPZ004777 is serially diluted

7、3-fold in DMSO for1/3 Master of Small Molecules 您邊的抑制劑師www.MedChemEa total of ten concentrations, beginning at 1 M. A 1 L aliquot of each inhibitor dilution is plated in a 384-wellmicrotiter plate. The 100% inhibition control consisted of 2.5 M final concentration of the product inhibitor S-adenosyl

8、-L-homocysteine, (SAH). Compound is incubated for 30 min with 40 L per well of 0.25 nMDOT1L(1-416) in assay buffer (20 mM TRIS pH 8.0 10 mM NaCl, 0.002% Tween 20, 0.005% Bovine SkinGelatin, 100 mM KCl, and 0.5 mM DTT). 10 L per well of substrate mix comprising assay buffer with 200 nM3H-SAM (80 Ci/m

9、mol), 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 formatreferred to as “balanced conditions”. Reactions are incubated for 120 min and quenched with 10 L per wellof 800

10、 M SAM. Incorporation of radioactivity into nucleosome substrate is measured in a flashplate. IC50values for enzymes in the histone methyltransferase panel are determined under similar balanced assayconditions with both SAM and protein/peptide substrate present at concentrations equal to their respe

11、ctiveKM values 1.MCE has not independently confirmed the accuracy of these methods. They are for reference only.Cell Assay 1 For assessment of cell proliferation and viability in human cell lines, exponentially growing cells are plated, intriplicate, in 96-well plates at a density of 3104 cells/well

12、 in a final volume of 150 L. Cells are incubated inthe presence of 3 M (proliferation curve), or increasing concentrations (IC50 determination) of EPZ004777up to 50 M. Viable cell number is determined every 3-4 days for up to 18 days using the Guava Viacountassay and analyzed on a Guava EasyCyte Plu

13、s instrument. On days of cell counts, growth media andEPZ004777 are replaced and cells split back to a density of 5104 cells/well. Total cell number is expressedas split-adjusted viable cells per well. For each cell line, IC50 values are determined from concentration-dependence curves at each time p

14、oint using Graphpad Prism software. Experiments to determine IC50values 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 Mice 1Administration 1 Nine

15、-week-old female nude mice (nu/nu) are injected subcutaneously with MV4-11 cells in the right flank(200 L of a 5107 cells/mL suspension in a 1:1 mixture of PBS and Matrigel). Mice are randomized totreatment groups when tumor sizes reached 300-400 mm3. Six mice received subcutaneous implant ofosmotic

16、 pumps, containing 50 mg/mL EPZ004777 in 10% ethanol, 90% water, and five control mice receivedno pump implant. Six days after pump implant, animals are sacrificed and tumor samples from treated andcontrol animals are collected for immunoblot analysis. For the disseminated leukemia model, MV4-11 cel

17、lsare transduced with the pMMP-LucNeo retrovirus. Eight-week-old female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ(NSG) mice are purchased from Jackson Laboratories. A total of 1107 MV4-11-LucNeo cells are injectedintravenously via the lateral tail vein. Engraftment of disseminate leukemia is determined by bio

18、luminescenceimaging after injection of 75 mg/kg of D-luciferin. Animals with documented leukemia are divided intotreatment groups consisting of vehicle (15% ethanol, 50% PEG300, 35% water) loaded osmotic pumps, orEPZ004777 at 50, 100, or 150 mg/mL. Osmotic pumps are replaced after one week. Irritati

19、on caused bycompound precipitation is observed in the 100 and 150 mg/mL dose groups, precluding additional pumpreplacements. Animals are monitored daily for clinical symptoms, and are euthanized when they displayedsigns of distress consistent with terminal leukemic disease. Log-rank analysis is used

20、 to determine statisticalsignificance of the survival curves.MCE has not independently confirmed the accuracy of these methods. They are for reference only.2/3 Master of Small Molecules 您邊的抑制劑師www.MedChemE戶使本產(chǎn)品發(fā)表的科研獻 Cell Syst. 2018 Apr 25;6(4):424-443.e7. Patent. US20180263995A1.See more customer validations on HYPERLINK / www.MedChemEREFERENCES1. Daigle SR, et al. Selective killing of mixed l