PP-121 is a multi-targeted inhibitor of PDGFR (IC50: 2 nM), Hck (IC50: 8 nM), mTOR (IC50: 10 nM), VEGFR2(IC50: 12 nM), Src (IC50: 14 nM) and Abl (IC50: 18 nM) , also inhibits DNA-PK (IC50: 60 nM).

Src:14 nM, PDGFR:2 nM, Hck:8 nM, mTOR:10 nM, Abl:18 nM, VEGFR2/KDR:12 nM

In glioma cell lines (U87 and LN229), PP-121 (0.04-10 μM) dose-dependently inhibited Akt, p70S6K and S6 phosphorylation, and PP-121 (0.04-10 μM) inhibited the proliferation of a range of tumor cells by directly inhibiting PI3Ks and mTOR. PP-121 induced cell arrest at G0/G1 phase in LN220, U87 and Seg1 cells. PP-121 (0.08-20 μM) inhibited v-Src-induced tyrosine phosphorylation in NIH3T3 cells transformed with v-Src (Thr338). Acting on NIH3T3 cells transformed with v-Src(Thr338), PP-121 (2.5 μM) restored the staining of actin fibers. PP-121 (40 nM) inhibited Ret autophosphorylation when applied to TT thyroid cancer cells expressing Ret mutation 35 of the C634W oncogene.PP-121 inhibited the proliferation of TT thyroid cancer cells (IC50: 50 nM). When acting on human umbilical vein endothelial cells, PP-121 inhibited VEGF-stimulated cell proliferation (IC50: 41 nM).

Kinase assays: Purified kinase domains are incubated with PP-121 at 2- or 4-fold dilutions over a concentration range of 1 nM-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.(Only for Reference)