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

作用于胶质瘤细胞系（U87和LN229）时,PP-121（0.04-10 μM）可剂量依赖性地有效抑制Akt、p70S6K 和S6磷酸化。PP-121（0.04-10 μM）通过直接抑制PI3Ks 和 mTOR,而有效抑制一系列肿瘤细胞增殖。作用于LN220,U87和Seg1细胞时,PP-121可诱导细胞在G0/G1期停滞。作用于转化v-Src(Thr338)的NIH3T3 细胞,PP-121（0.08-20 μM）抑制v-Src诱导的酪氨酸磷酸化。作用于转化v-Src(Thr338)的NIH3T3细胞时,PP-121（2.5 μM）使肌动蛋白纤维的染色恢复。作用于表达C634W致癌基因 Ret突变35的TT甲状腺癌细胞时,PP-121（40 nM）抑制Ret自磷酸化。PP-121抑制 TT 甲状腺癌细胞增殖（IC50：50 nM）。作用于人脐静脉内皮细胞时,PP-121抑制VEGF刺激的细胞增殖（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)