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

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)