OSU-03012 (AR-12)

PDK-1 (IC50 = 5 μM)

OSU-03012 induces apoptotic death in PC-3 cells with IC50 of 5 µM and reduces the activity of immunoprecipitated p70S6K. OSU-03012 completely inhibits cell growth in a variety of tumor cell lines at concentrations as low as 3-5 m, versus the concentration of at least 50 m needed for celecoxib.[1] Compared to untransformed astrocytes, OSU-03012 more strongly encourages cell killing in glioma cells. OSU-03012 induces cell death in a dose-dependent manner that is unaffected by the p53 mutation, the expression of ERBB1 VIII, or the loss of phosphatase and tensin function brought on by a homolog deletion on chromosome 10. Ionizing radiation and OSU-03012 increase cell death in an additive, caspase-independent manner. OSU-03012 lethality as a single agent or when combined with signaling modulators is not modified in cells lacking expression of BIM or of BAX/BAK. OSU-03012 encourages the release of cathepsin B from the lysosomal compartment and AIF from the mitochondria. In protein kinase R-like endoplasmic reticulum kinase-/- cells, the lethality of OSU-03012 is reduced, which is correlated with decreased BID cleavage and reduced cathepsin B and AIF release into the cytosol. [2] OSU-03012 inhibits the growth, migration, and apoptosis of thyroid cancer cells (NPA, WRO, and ARO cells), which causes a rise in S phase cells without a rise in G2 cells. OSU-03012 is an ATP-competitive inhibitor of PAK activity that prevents thyroid cancer cells from phosphorylating AKT. [3] With IC50 values under 1 M, OSU-03012 inhibits the growth of Huh7, Hep3B, and HepG2 cell lines, which are used to study hepatocellular carcinoma. In Huh7 cells, OSU-03012 induces autophagy but does not suppress PDK1 or AKT activity or cause cellular apoptosis. After OSU-03012 treatment, accumulation of reactive oxygen species (ROS) is also found. [4] According to a recent study, OSU-03012 may make (Bcr)-Abl mutant cell lines more susceptible to apoptosis caused by imatinib. [5]

OSU-03012 suppresses tumor growth by 57.59% and increases cleaved LC3 in Huh7 tumor xenografts at 200 mg/kg. [4] OSU-03012 significantly reduces EGFR protein expression in tumors by 48% when compared to vehicle controls and inhibits YB-1 from binding to the EGFR promoter in MDA-MB-435/LCC6 xenografts. [6] The oral administration of OSU-03012 results in a 55% growth inhibition of HMS-97 schwannoma xenografts and is well tolerated. [7]

A PDK-1 kinase assay kit is used in this in vitro test. This cell-free assay is based on recombinant PDK-1's capacity to activate its downstream serum- and glucocorticoid-regulated kinase in the presence of DMSO vehicle or OSU-03012, which in turn phosphorylates the Akt/serum- and glucocorticoid-regulated kinase-specific peptide substrate RPRAATF with [γ-32P]ATP. Using P81 phosphocellulose paper and three washes with 0.75% phosphoric acid, the 32P-phosphorylated peptide substrate is then separated from the remaining [γ-32P]-ATP. The quantity is then measured in a scintillation counter.

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. In 96-well, flat-bottomed plates, cells are grown for 24 hours in RPMI 1640 with 10% FBS supplement. They are exposed to different concentrations of OSU-03012 (0-10 μM) dissolved in DMSO (final concentration≤0.1%) in 1% serum–containing RPMI 1640 for various lengths of time (–72 hours). At a level comparable to that in OSU-03012-treated cells, controls are given DMSO vehicle. In place of the medium, 200 L of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide in 10% FBS-containing RPMI 1640 are added. The cells are incubated for two hours at 37 °C in the CO2 incubator. The reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide dye is solubilized in 200 L DMSO per well after the supernatants are removed from the wells. Absorbance at 570 nm is determined by using a plate reader.

Mice[3]
SCID/Rag2m mice (6-8 weeks old, female) are subcutaneously injected with 1×107 MDA-MB-435/LCC6 cells stably transfected with HER-2/neu. The right and left sides of the lower back of each mouse are injected with the cells. We inject two tumors into each of the eight mice. The mice are randomly divided into the vehicle, 0.5% methyl cellulose/0.1% Tween 80, or OSU-03012 groups after six weeks. The vehicle or OSU-03012 are administered orally to mice once daily for three days. On the fourth day of the experiment, mice are killed, the tumors are removed, and chromatin immunoprecipitation (ChIP) and protein isolations are performed on the tumors.

[1]. Oncotarget . 2015 Aug 21;6(24):20570-7.

[2]. Mol Cancer Res . 2014 May;12(5):803-12.

C26H19F3N4O

460.4505

460.1511

C, 67.82; H, 4.16; F, 12.38; N, 12.17; O, 3.47

742112-33-0

742112-33-0;1471979-81-3 (HCl);

Solid powder

C1=CC=C2C(=C1)C=CC3=C2C=CC(=C3)C4=CC(=NN4C5=CC=C(C=C5)NC(=O)CN)C(F)(F)F

YULUCECVQOCQFQ-UHFFFAOYSA-N

InChI=1S/C26H19F3N4O/c27-26(28,29)24-14-23(33(32-24)20-10-8-19(9-11-20)31-25(34)15-30)18-7-12-22-17(13-18)6-5-16-3-1-2-4-21(16)22/h1-14H,15,30H2,(H,31,34)

2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl} acetamide

AR12; AR 12; AR-12; OSU-03012; OSU03012; OSU 03012

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~11 mg/mL (~23.9 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

0.5% methylcellulose+0.2% Tween 80: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.)