319.36 PP-121 is a multi-targeted inhibitor of PDGFR, Hck, mTOR, VEGFR2, Src and Abl with IC50 of 2 nM, 8 nM, 10 nM, 12 nM, 14 nM and 18 nM, also inhibits DNA-PK with IC50 of 60 nM.
PP-121 selectivity interacts within a hydrophobic pocket that is
conserved between both tyrosine kinases and PI3Ks, not serine-threonine
kinases. PP-121 mak
es a hydrogen bond to Glu310 in Src, effectively
substituting for the structural role of the catalytic lysine and
resulting in the ordering of helix C and stabilization of an active
conformation. PP-121 also inhibits other PI3Ks including p110α and
DNA-PK with IC50 of 52 nM and 60 nM, respectively. PP-121 potently and
dose-dependently blocks the phosphorylation of Akt, p70S6K and S6 in two
glioblastoma cell lines, U87 and LN229.
PP-121 potently inhibits the
proliferation of a subset of the tumor cell lines by direct inhibition
of PI3Ks and mTOR. PP-121 induces a G0/G1 arrest in LN220, U87 and Seg1
cells. PP-121 also blocks tyrosine phosphorylation induced by v-Src in
NIH3T3 cells transformed with v-Src(Thr338). PP-121 could restore actin
stress fiber staining in NIH3T3 cells transformed with v-Src(Thr338).
PP-121 at a low concentration of 40 nM inhibits Ret autophosphorylation
in TT thyroid carcinoma cells that express the C634W oncogenic Ret
mutant35. PP-121 inhibits cell proliferation with IC50 of 50 nM in TT
thyroid carcinoma cells. PP-121 inhibits cell proliferating stimulated
only with VEGF with IC50 of 41 nM in human umbilical vein endothelial
cells (HUVECs). PP-121 directly inhibits Bcr-Abl induced tyrosine
phosphorylation, resulting in drug-induced Apoptosis in K562 cells and a
combination of apoptosis and Cell Cycle arrest in Bcr-Abl expressing
Purified kinase domains are incubated with PP-121 at 2- or 4-fold
dilutions over a concentration range of 1nM-50 µM or with vehicle (0.1%
DMSO) in the presence of 10 µM ATP, 2.5 µCi of γ-32P-ATP and
substrate. Reactions are terminated by spotting onto nitrocellulose or
phosphocellulose membranes, depending on the substrate; this membrane is
then washed 5–6 times to remove unbound radioactivity and dried.
Transferred radioactivity is quantitated by phosphorimaging and IC50
values are calculated by fitting the data to a sigmoidal doseresponse
using Prism software.
For western blot analysis, cells are grown in 12-well plates and treated
with PP-121 at the indicated concentrations or vehicle (0.1% DMSO).
Treated cells are lysed, lysates are resolved by SDS-PAGE, transferred
to nitrocellulose and blotted. For cell proliferation assays,cells are
grown in 96-well plates are treated with PP-121 at 4-fold dilutions (10
µM - 0.040 μM) or vehicle (0.1% DMSO).
After 72 hours cells are exposed
to Resazurin sodium salt (22 µM) and fluorescence is quantified. IC50
values are calculated using Prism software. For proliferation assays
involving single cell counting, non-adherent cells are plated at low
density (3–5% confluence) and treated with PP-121 (2.5 µM) or vehicle
(0.1% DMSO). Cells are diluted into trypan blue daily and viable cells
counted using a hemocytometer.
For apoptosis and cell cycle analysis,
cells are treated with the indicated concentration of PP-121 or vehicle
(0.1% DMSO) for 24–72 hours. Cells are either stained live with
AnnexinV-FITC or fixed with ethanol and stained with propidium iodide.
Cell populations are separated using a FacsCalibur flow cytometer; data
is collected using CellQuest Pro software and analyzed with either
ModFit or FlowJo Software.
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