OSU-03012 (AR-12)

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Biological Activity

Description	OSU-03012 is a potent inhibitor of recombinant PDK-1 with IC50 of 5 μM and 2-fold increase in potency over OSU-02067.
Targets	PDK-1
IC50	5 μM [1]
In vitro	OSU-03012 induces apoptotic death in PC-3 cells with IC50 of 5 µM and reduces the activity of immunoprecipitated p70S6K. OSU-03012 completely suppress cell growth in a diverse range of tumor cell lines at concentrations of 3–5 μm, as compared with the concentration of at least 50 μm required for celecoxib.[1] OSU-03012 promotes cell killing to a greater extent in glioma cells than in nontransformed astrocytes. OSU-03012 causes a dose-dependent induction of cell death that is not altered by p53 mutation, expression of ERBB1 VIII, or loss of phosphatase and tensin function due to a homolog deletion on chromosome 10. OSU-03012 and ionizing radiation cause an additive, caspase-independent elevation in cell killing. 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 promotes the release of cathepsin B from the lysosomal compartment and that of AIF from mitochondria. The lethality of OSU-03012 is attenuated in protein kinase R-like endoplasmic reticulum kinase-/- cells, which correlated with the reduced cleavage of BID and suppression of cathepsin B and AIF release into the cytosol. [2] OSU-03012 inhibits thyroid cancer cell (NPA, WRO, and ARO cells) proliferation, migration and induces apoptosis, which results in an increase of cells in the S phase without an increase of cells in G2. OSU-03012 is an ATP-competitive inhibitor of PAK activity and suppresses the phosphorylation of AKT in thyroid cancer cells. [3] OSU-03012 inhibits cell growth of hepatocellular carcinoma cell lines including Huh7, Hep3B and HepG2 cells with IC50 values below 1 μM. OSU-03012 does not suppress PDK1 or AKT activity or induce cellular apoptosis but induces autophagy in Huh7 cells. Moreover, accumulation of reactive oxygen species (ROS) is detected after OSU-03012 treatment. [4] A recent study shows that OSU-03012 could enhance the susceptibility of (Bcr)-Abl mutant cell lines to imatinib-induced apoptosis. [5]
In vivo	OSU-03012 suppresses tumor growth by 57.59% and increases cleaved LC3 in Huh7 tumor xenografts at 200 mg/kg. [4] OSU-03012 remarkably decreases expression of EGFR protein in the tumors by 48% compared with vehicle controls and also prevents YB-1 from binding to the EGFR promoter in MDA-MB-435/LCC6 xenografts.[6] OSU-03012 is well tolerated and inhibits the growth of HMS-97 schwannoma xenografts by 55% after oral administration. [7]
Features	A derivative of celecoxib with 10-fold greater antitumor activity, but lacks celecoxibs COX-2 inhibitory activity.

Protocol(Only for Reference)

Kinase Assay: ^[1]

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.

Cell Assay: ^[1]

Cell lines	PC-3 cells
Concentrations	0-10 μM
Incubation Time	~72 hours
Method	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.

Animal Study: ^[4]

Animal Models	Huh7 tumor xenografts in male BALB/c nude mice
Dosages	100-200 mg/kg
Administration	Daily by gavage
Solubility	0.5% methylcellulose/0.2% Tween 80, 30 mg/mL

1

References

Chemical Information

Download OSU-03012 (AR-12) SDF

Chemical Name	2-amino-N-(4-(5-(phenanthren-2-yl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)acetamide

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.46045	4.6045	9.209	-

Customer Reviews (1)

Click to enlarge	Rating
	Source	PLoS One, 2013, 8(9):e75100 . OSU-03012 (AR-12) purchased from Selleck
	Method	Western blot
	Cell Lines	Orbital fibroblasts
	Concentrations	5 uM
	Incubation Time	6 h
	Results	The siRNA targeting PDK1 attenuates TSH-dependent PKCm Ser 916 phosphorylation in fibroblasts (Fig. C) while the PDK1 inhibitor, OSU-03012 (5 mM) [54] blocks TSH-dependent phosphorylation of PKCm Ser 916 and PKCbII Ser 660 levels in the respective cell-types (Fig. C). The inhibitor also reduces levels of TSH-dependent pAKT Threonine 308 in fibroblasts but fails to do so in fibrocytes (Fig. D, 14.461.2% and 2.560.6%, respectively).

Product Citations (4)

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  Thyrotropin Regulates IL-6 Expression in CD34+ Fibrocytes: Clear Delineation of Its cAMP-Independent Actions. [Raychaudhuri N, et al. PLoS One 2013;8(9):e75100]

  
  PubMed: 24086448


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  Endogenous adenosine contributes to renal sympathetic neurotransmission via postjunctional A1 receptor-mediated coincident signaling. [Edwin K, et al. Am J Physiol Renal Physiol 2011;302(4):F466-76]

  
  PubMed: 22114202


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  Cryptococcus neoformans Phosphoinositide-Dependent Kinase 1 (PDK1) Ortholog Is Required for Stress Tolerance and Survival in Murine Phagocytes. [Chabrier-Roselló Y, et al. Eukaryot Cell 2012;12(1):12-22]

  
  PubMed: 23087368