GSK343 1346704-33-3

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Molecular Weight:

541.69 GSK343 is a potent and Selective EZH2 Inhibitor with IC50 of 4 nM, showing 60 fold selectivity against EZH1, and >1000 fold selectivity against other histone methyltransferases.


Biological Activity


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. 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.






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.

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

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Product Categories : Epigenetics > Histone Methyltransferase Inhibitor