EI1

Catalog No.S7611 Batch:S761101

Technical Data

Formula

C₂₃H₂₆N₄O₂

Molecular Weight

390.48

CAS No.

1418308-27-6

Solubility (25°C)*

In vitro

DMSO

42 mg/mL (107.55 mM)

Water

Insoluble

Ethanol

Insoluble

In vivo (Add solvents to the product individually and in order)

Clear solution	5% DMSO 1 95% Corn oil	0.26mg/ml	Taking the 1 mL working solution as an example, add 50 μL of 5.2 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.
Clear solution	5%DMSO 1 40%PEG300 2 5%Tween80 3 50%ddH2O	0.26mg/ml	Taking the 1 mL working solution as an example, add 50 μL of 5.2 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 clarify; 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.

* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
* Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

Preparing Stock Solutions

Biological Activity

Description

EI1 is a potent and selective EZH2 inhibitor with IC50 of 15 nM and 13 nM for EZH2 (WT) and EZH2 (Y641F), respectively.

Targets

EZH2 (Y641F) ^[1] (Cell-free assay)	Ezh2 (wild-type) ^[1] (Cell-free assay)
13 nM	15 nM

In vitro

In DLBCL cells, EI1 inhibits cellular H3K27 methylation and activates Ezh2 target gene p16 expression. In mouse embryonic fibroblasts, EI1 also inhibit H3K27me3 and cell proliferation. In addition, EI1 selectively inhibits the growth of DLBCL cells carrying Ezh2 mutation, and causes cell cycle arrest and apoptosis. ^[1]

Protocol (from reference)

Kinase Assay:^{^[1]}	Biochemical Assay For IC50 determination, EI1 is serial diluted threefold in DMSO for a total of 12 concentrations, with the starting concentration at 1 μM. The reaction is incubated at room temperature for 120 min, and stopped by adding quench solution (2.5% TFA with 320 nM d4-SAH). SAH production is quantitated using an API 4000 triple quadrupole mass spectrometry with Turbulon Spray coupled with Prominence UFLC. The percentage of inhibition is normalized using positive (no inhibitor) and negative (no enzyme) controls, and IC50 calculated using PRISM. Enzymology studies of S-Adenosyl methionine (SAM) competition are performed with slight modification of reaction condition: 10 μM EI1 is used as the starting dose for serial dilution. SAM is titrated over a range between 1 μM and 50 μM (corresponding to 1 × Km and 50 × Km), and substrate peptide is present in the final reaction mixture at its saturated condition (10 μM). For histone methyltransferase (HMT) profiling in Table 1, all HMTs are purified recombinant proteins from either Escherichia coli or Baculovirus system. The catalytic domain of G9a, SuV39H2, Set7/9, CARM1, SETD8, NSD3, SETD2, and Dot1L, and the full-length SmyD2 protein were used in the biochemical assays. HMT biochemical reactions are carefully characterized with enzymology studies and the SAM and substrate Km determined. The SAM and substrate concentrations are kept at their respective Km for most of the HMTs, except the ones (SmyD2 and Set7/9) with low SAM-Km value, for which 0.5 μM SAM is used. All HMT reactions are performed using the same assay format where the production of SAH from the biochemical reaction is quantitated by LC-MS.
Cell Assay:^{^[1]}	Cell lines Ezh2 mutant DLBCL cells (WSU-DLCL2, SU-DHL6, Karpas422, DB, and SU-DHL4), Ezh2 wild-type DLBCL cells (OCI-LY19 and GA10), and Mouse embryonic fibroblasts (MEFs) Concentrations ~10 μM Incubation Time 11-15 days Method Exponentially growing diffused large B-cell lymphoma (DLBCL) cells are seeded in 12-well plates at a density of 1 × 10⁵ cells/mL with the indicated concentration of EI1. Viable cell number is determined every 3–4 d for up to 14 or 15 d by Vi-CELL. Mouse embryonic fibroblasts (MEFs) are seeded in a six-well plate at 2.5 × 10⁴ cell/mL and treated with EI1 (3.3 μM) or 4-OH-tamoxifen (100 nM). Viable cell number is determined at days 3, 6 and 11. On days of cell counts, fresh growth medium and compound are replenished and cells split back to a density of 1 × 10⁵ cells/mL. Total cell number is expressed as split-adjusted viable cells per milliliter. IC50 is calculated by PRISM and all proliferation experiments are repeated more than two times and representative data are presented.

Kinase Assay:^{^[1]}

Biochemical Assay
For IC50 determination, EI1 is serial diluted threefold in DMSO for a total of 12 concentrations, with the starting concentration at 1 μM. The reaction is incubated at room temperature for 120 min, and stopped by adding quench solution (2.5% TFA with 320 nM d4-SAH). SAH production is quantitated using an API 4000 triple quadrupole mass spectrometry with Turbulon Spray coupled with Prominence UFLC. The percentage of inhibition is normalized using positive (no inhibitor) and negative (no enzyme) controls, and IC50 calculated using PRISM. Enzymology studies of S-Adenosyl methionine (SAM) competition are performed with slight modification of reaction condition: 10 μM EI1 is used as the starting dose for serial dilution. SAM is titrated over a range between 1 μM and 50 μM (corresponding to 1 × Km and 50 × Km), and substrate peptide is present in the final reaction mixture at its saturated condition (10 μM). For histone methyltransferase (HMT) profiling in Table 1, all HMTs are purified recombinant proteins from either Escherichia coli or Baculovirus system. The catalytic domain of G9a, SuV39H2, Set7/9, CARM1, SETD8, NSD3, SETD2, and Dot1L, and the full-length SmyD2 protein were used in the biochemical assays. HMT biochemical reactions are carefully characterized with enzymology studies and the SAM and substrate Km determined. The SAM and substrate concentrations are kept at their respective Km for most of the HMTs, except the ones (SmyD2 and Set7/9) with low SAM-Km value, for which 0.5 μM SAM is used. All HMT reactions are performed using the same assay format where the production of SAH from the biochemical reaction is quantitated by LC-MS.

Cell Assay:^{^[1]}

Cell lines
Ezh2 mutant DLBCL cells (WSU-DLCL2, SU-DHL6, Karpas422, DB, and SU-DHL4), Ezh2 wild-type DLBCL cells (OCI-LY19 and GA10), and Mouse embryonic fibroblasts (MEFs)
Concentrations
~10 μM
Incubation Time
11-15 days
Method
Exponentially growing diffused large B-cell lymphoma (DLBCL) cells are seeded in 12-well plates at a density of 1 × 10⁵ cells/mL with the indicated concentration of EI1. Viable cell number is determined every 3–4 d for up to 14 or 15 d by Vi-CELL. Mouse embryonic fibroblasts (MEFs) are seeded in a six-well plate at 2.5 × 10⁴ cell/mL and treated with EI1 (3.3 μM) or 4-OH-tamoxifen (100 nM). Viable cell number is determined at days 3, 6 and 11. On days of cell counts, fresh growth medium and compound are replenished and cells split back to a density of 1 × 10⁵ cells/mL. Total cell number is expressed as split-adjusted viable cells per milliliter. IC50 is calculated by PRISM and all proliferation experiments are repeated more than two times and representative data are presented.

References

[1] Qi W, et al. Proc Natl Acad Sci U S A. 2012, 109(52), 21360-21365.

Customer Product Validation

: , , Oncotarget, 2015, 6(32):32410-25.

: , , Mol Cancer, 2017, 16(1):5

: Data from [Data independently produced by , , Oncotarget, 2015, 6(32): 32410-32425.]

Selleck's EI1 has been cited by 4 publications

Epigenetic Reprogramming with Antisense Oligonucleotides Enhances the Effectiveness of Androgen Receptor Inhibition in Castration-Resistant Prostate Cancer [ Cancer Res, 2018, 78(20):5731-5740]	PubMed: 30135193
Long non-coding RNA TUG1 is involved in cell growth and chemoresistance of small cell lung cancer by regulating LIMK2b via EZH2. [Niu Y, et al. Mol Cancer, 2017, 10.1186/s12943-016-0575-6]	PubMed: 28069000
The Human Immunodeficiency Virus 1 ASP RNA promotes viral latency by recruiting the Polycomb Repressor Complex 2 and promoting nucleosome assembly. [ Virology, 2017, 506:34-44]	PubMed: 28340355
NBAT1 suppresses breast cancer metastasis by regulating DKK1 via PRC2 [Hu P, et al. Oncotarget, 2015, 6(32):32410-25]	PubMed: 26378045

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SHIPPING AND STORAGE
Selleck products are transported at room temperature. If you receive the product at room temperature, please rest assured, the Selleck Quality Inspection Department has conducted experiments to verify that the normal temperature placement of one month will not affect the biological activity of powder products. After collecting, please store the product according to the requirements described in the datasheet. Most Selleck products are stable under the recommended conditions.

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