GSK343

Catalog No.S7164 Batch:S716405

Technical Data

Formula	C₃₁H₃₉N₇O₂
Molecular Weight	541.69	CAS No.	1346704-33-3
Solubility (25°C)*	In vitro	DMSO	100 mg/mL (184.6 mM)
		Ethanol	12 mg/mL (22.15 mM)
		Water	Insoluble
* <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.)

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Biological Activity

Description

GSK343 is a potent and selective EZH2 inhibitor with IC50 of 4 nM in a cell-free assay, showing 60 fold selectivity against EZH1, and >1000 fold selectivity against other histone methyltransferases. GSK343 induces autophagy.

Targets

EZH2 ^[1] (Cell-free assay)	EZH1 ^[1] (Cell-free assay)
4 nM	240 nM

In vitro

GSK343 inhibits trimethylation of H3K27 (H3K27me3) with IC50 of 174 nM in HCC1806 breast cancer cells. GSK343 potently inhibits cell proliferation in breast cancer cells and prostate cancer cells, and the prostate cancer cell line LNCaP is the most sensitive to GSK343, with IC50 of 2.9 μM. ^[1]

GSK343 significantly suppresses the growth of EOC cells cultured in 3D matrigel extracellular matrix (ECM), which mimics the tumor microenvironment in vivo. In addition, GSK343 also induces apoptosis of EOC cells in 3D and significantly inhibits the invasion of EOC cells. ^[2]

In vivo

GSK343, a selective EZH2 inhibitor, inhibits phagocytosis.

Features

A chemical probe for the SGC epigenetics initiative. Potential use in a variety of solid tumors.

Protocol (from reference)

Kinase Assay:^{^[1]}	In vitro biochemical assays against histone methyltransferases Activity against EZH2 is assessed using 5 member PRC2 complex (Flag-EZH2, EED, SUZ12, AEBP2, RbAp48). The assay protocol may be summarized as follows: 10 mM stocks of compounds are prepared from solid in 100% DMSO. An 11 point serial dilution master plate is prepared in 384 well format (1:3 dilution, columns 6 and 18 were equal volume DMSO controls) and dispensed to assay ready plates using acoustic dispensing technology to create a 100 nL stamp of compound and DMSO controls. The assay additions consisted of equal volume additions of 10 nM EZH2 and the substrate solution (5 µg/mL HeLa nucleosomes and 0.25 µM [³H]-SAM) dispensed into assay plates using a multi-drop combi dispense. Reaction plates are incubated for 1 hr and quenched with an equal volume addition of 0.5 mg/mL PS-PEI Imaging Beads (RPNQ0098) containing 0.1 mM unlabeled SAM. The plates are sealed, dark adapted for 30 minutes, and a 5 minute endpoint luminescence image is acquired using a Viewlux imager. Plate statistics such as Z’ and signal to background as well as dose response curves are analyzed using Activity BaseXE. The in vitro biochemical activity of EZH1 is assessed as part of a 5 member PRC2 complex using a 384 well SPA assay identical to EZH2. Buffer components, reagent dispensing, compound plate preparation, quench conditions and data analysis are identical for EZH1 and EZH2 with final assay concentrations of 20 nM EZH1, 5 μM/mL HeLa nucleosomes and 0.25 μM [³H]-SAM. Further data analysis, pIC50 pivots and visualizations are enabled by TIBCO Spotfire. Compounds are profiled at Reaction Biology Corp. (Malvern, PA) to assess inhibition in their panel of histone methyltransferase assays. Methyltransferase activity is assessed using HotSpot technology, a miniaturized radioisotope-based filter binding assay. Inhibitors are dissolved in dimethyl sulfoxide (DMSO) and tested at concentrations up to 100 uM with a final DMSO concentration of 2%. Buffer containing the methyltrasferase at the listed concentration and its preferred substrate as shown in the accompanying table is preincubated with compound for 10 min. Reactions are initiated by the addition of 1 uM S-adenosyl-L-[methyl-³H]methionine (SAM), allowed to incubate for 60 min at 30C followed by transfer to P81 filter-paper and PBS wash before detection.
Cell Assay:^{^[1]}	Cell lines Breast cancer cell lines (HCC1806, Sk-Br-3, ZR-75-1), prostate cancer cell lines (DU145, PC3, LNCaP) Concentrations ~50 μM Incubation Time 6 days Method To account for varying doubling rates among cancer cell lines, the optimal cell seeding is determined empirically for all cell lines by examining their growth in a 384-well plate over 6 days with a wide range of seeding densities. Cells are then plated at the optimal seeding density and allowed to adhere overnight. Cells are treated in duplicate with a 20-point 2-fold dilution series of compound or 0.147% DMSO (vehicle control) and incubated for 6 days at 37C in 5% CO2. Cells are then lysed with 25 μl CellTiter-Glo per well and chemiluminescence is quantified with a TECAN Safire2 microplate reader. In addition, an untreated plate of cells is harvested at the time of compound addition (T0) to quantify the starting number of cells. CTG values after 6 days of treatment were expressed as a percent of the T0 value and plotted against compound concentration. Data are fit with a 4-parameter equation to generate a concentration response curve and the concentration of compound required to inhibit 50% of growth (gIC50) is determined

Kinase Assay:^{^[1]}

In vitro biochemical assays against histone methyltransferases
Activity against EZH2 is assessed using 5 member PRC2 complex (Flag-EZH2, EED, SUZ12, AEBP2, RbAp48). The assay protocol may be summarized as follows: 10 mM stocks of compounds are prepared from solid in 100% DMSO. An 11 point serial dilution master plate is prepared in 384 well format (1:3 dilution, columns 6 and 18 were equal volume DMSO controls) and dispensed to assay ready plates using acoustic dispensing technology to create a 100 nL stamp of compound and DMSO controls. The assay additions consisted of equal volume additions of 10 nM EZH2 and the substrate solution (5 µg/mL HeLa nucleosomes and 0.25 µM [³H]-SAM) dispensed into assay plates using a multi-drop combi dispense. Reaction plates are incubated for 1 hr and quenched with an equal volume addition of 0.5 mg/mL PS-PEI Imaging Beads (RPNQ0098) containing 0.1 mM unlabeled SAM. The plates are sealed, dark adapted for 30 minutes, and a 5 minute endpoint luminescence image is acquired using a Viewlux imager. Plate statistics such as Z’ and signal to background as well as dose response curves are analyzed using Activity BaseXE. The in vitro biochemical activity of EZH1 is assessed as part of a 5 member PRC2 complex using a 384 well SPA assay identical to EZH2. Buffer components, reagent dispensing, compound plate preparation, quench conditions and data analysis are identical for EZH1 and EZH2 with final assay concentrations of 20 nM EZH1, 5 μM/mL HeLa nucleosomes and 0.25 μM [³H]-SAM. Further data analysis, pIC50 pivots and visualizations are enabled by TIBCO Spotfire. Compounds are profiled at Reaction Biology Corp. (Malvern, PA) to assess inhibition in their panel of histone methyltransferase assays. Methyltransferase activity is assessed using HotSpot technology, a miniaturized radioisotope-based filter binding assay. Inhibitors are dissolved in dimethyl sulfoxide (DMSO) and tested at concentrations up to 100 uM with a final DMSO concentration of 2%. Buffer containing the methyltrasferase at the listed concentration and its preferred substrate as shown in the accompanying table is preincubated with compound for 10 min. Reactions are initiated by the addition of 1 uM S-adenosyl-L-[methyl-³H]methionine (SAM), allowed to incubate for 60 min at 30C followed by transfer to P81 filter-paper and PBS wash before detection.

Cell Assay:^{^[1]}

Cell lines
Breast cancer cell lines (HCC1806, Sk-Br-3, ZR-75-1), prostate cancer cell lines (DU145, PC3, LNCaP)
Concentrations
~50 μM
Incubation Time
6 days
Method
To account for varying doubling rates among cancer cell lines, the optimal cell seeding is determined empirically for all cell lines by examining their growth in a 384-well plate over 6 days with a wide range of seeding densities. Cells are then plated at the optimal seeding density and allowed to adhere overnight. Cells are treated in duplicate with a 20-point 2-fold dilution series of compound or 0.147% DMSO (vehicle control) and incubated for 6 days at 37C in 5% CO2. Cells are then lysed with 25 μl CellTiter-Glo per well and chemiluminescence is quantified with a TECAN Safire2 microplate reader. In addition, an untreated plate of cells is harvested at the time of compound addition (T0) to quantify the starting number of cells. CTG values after 6 days of treatment were expressed as a percent of the T0 value and plotted against compound concentration. Data are fit with a 4-parameter equation to generate a concentration response curve and the concentration of compound required to inhibit 50% of growth (gIC50) is determined

References

Customer Product Validation

: Data from [Data independently produced by , , Nucleic Acids Res, 2016, 44(16):7659-72.]

: Data from [Data independently produced by , , Oncogene, 2015, 10.1038/onc.2015.38]

: Data from [Data independently produced by , , Mol Cancer, 2017, 16(1):5.]

Selleck's GSK343 has been cited by 57 publications

LINC01021 Attenuates Expression and Affects Alternative Splicing of a Subset of p53-Regulated Genes [ Cancers (Basel), 2024, 16(9)1639]	PubMed: 38730591
Select EZH2 inhibitors enhance viral mimicry effects of DNMT inhibition through a mechanism involving NFAT:AP-1 signaling [ Sci Adv, 2024, 10(13):eadk4423]	PubMed: 38536911
Polycomb repressor complex 2 suppresses interferon-responsive MHC-II expression in melanoma cells and is associated with anti-PD-1 resistance [ J Immunother Cancer, 2023, 11(11)e007736]	PubMed: 38315170
Targeting polycomb repressor complex 2-mediated bivalent promoter epigenetic silencing of secreted frizzled-related protein 1 inhibits cholangiocarcinoma progression [ Clin Transl Med, 2023, 13(12):e1502]	PubMed: 38050190
H3K27me3 of Rnf19a promotes neuroinflammatory response during Japanese encephalitis virus infection [ J Neuroinflammation, 2023, 20(1):168]	PubMed: 37480121
H3K27me3 of Rnf19a promotes neuroinflammatory response during Japanese encephalitis virus infection [ J Neuroinflammation, 2023, 20(1):168]	PubMed: 37480121
Inhibition of EED activity enhances cell survival of female germline stem cell and improves the oocytes production during oogenesis in vitro [ Open Biol, 2023, 13(1):220211]	PubMed: 36695089
Polycomb-lamina antagonism partitions heterochromatin at the nuclear periphery [ Nat Commun, 2022, 13(1):4199]	PubMed: 35859152
Improving the repopulation capacity of elderly human hepatocytes by decoding aging-associated hepatocyte plasticity [ Hepatology, 2022, 10.1002/hep.32443]	PubMed: 35243665
Heterogeneity of tyrosine-based melanin anabolism regulates pulmonary and cerebral organotropic colonization microenvironment of melanoma cells [ Theranostics, 2022, 12(5):2063-2079]	PubMed: 35265199

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