Osu03012 (AR-12) is an orally bioavailable, small-molecule, celecoxib-derived inhibitor of phosphoinositide-dependent kinase-1 (PDK1) with potential antineoplastic activity.

PDK1:5 μM

在MDA-MB-435 / LCC6异种移植物模型中,OSU-03012显着降低肿瘤中EGFR蛋白的表达,同时还阻止YB-1与的EGFR启动子结合.在Huh7 移植瘤中,OSU-03012（200 mg/kg）能够抑制肿瘤细胞生长.在HMS-97神经鞘瘤异种移植物模型中,口服给药OSU-03012能够抑制细胞生长.

OSU-03012浓度为3-5 μM时即可完全抑制多种肿瘤细胞生长。在甲状腺癌细胞(NPA, WRO和ARO细胞)中,OSU-03012作为ATP竞争性抑制剂,通过 抑制PAK活性和AKT磷酸化,从而抑制细胞增殖和迁移,诱导细胞凋亡。在肝细胞癌细胞系（Huh7, Hep3B和HepG2细胞）中,OSU-03012（IC50<1 μM）抑制细胞生长。其中,对于Huh7细胞,OSU-03012诱导细胞发生自噬。在神经胶质瘤细胞、PC-3细胞中,OSU-03012能够促进细胞凋亡。

PDK-1 Kinase Assay: This in vitro assay is performed using a PDK-1 kinase assay kit. This cell-free assay is based on the ability of recombinant PDK-1, in the presence of DMSO vehicle or OSU-03012, to activate its downstream serum- and glucocorticoid-regulated kinase which, in turn, phosphorylates the Akt/serum- and glucocorticoid-regulated kinase-specific peptide substrate RPRAATF with [γ-32P]ATP. The 32P-phosphorylated peptide substrate is then separated from the residual [γ-32P]-ATP by using P81 phosphocellulose paper and quantitated in a scintillation counter after three washes with 0.75% phosphoric acid.

The effect of OSU-03012 on PC-3 cell viability is assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay in six replicates. Cells are grown in 10% FBS- supplemented RPMI 1640 in 96-well, flat-bottomed plates for 24 hours. They are exposed to various concentrations of OSU-03012 (0-10 μM) dissolved in DMSO (final concentration ≤0.1%) in 1% serum-containing RPMI 1640 for different time intervals (~72 hours). Controls receive DMSO vehicle at a concentration equal to that in OSU-03012-treated cells. The medium is removed and replaced by 200 μL of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide in 10% FBS-containing RPMI 1640. The cells are incubated in the CO2 incubator at 37 °C for 2 hours. Supernatants are removed from the wells, and the reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide dye is solubilized in 200 μL DMSO per well. Absorbance at 570 nm is determined by using a plate reader.(Only for Reference)