GSK 650394

Scintillation proximity assay (SPA)

SGK1 S422D (60-431 aa; 0.275 μg/mL final concentration) or SGK2 (0.875 μg/mL final concentration) were activated by PDK1 (1.1 μg/mL final concentration) in a buffer consisting of 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 mM EDTA, 10 mM MgCl2, 0.1% β-mercaptoethanol, 1 mg/mL BSA, and ATP (final concentration of 0.15 mM) and incubated for 30 mins at 30 °C. SGK2 was prepared exactly as described for SGK1, except it corresponded to the full-length protein. A solution containing biotinylated CROSStide peptide at a final concentration of 75 μM and γ32P-ATP corresponding to 2 × 106 cpm was prepared in the reaction buffer. In a 96-well plate, 5 μL of GSK 650394 was added to 25 μL of the activated enzyme mixture. To this, 20 μL of the CROSStide mixture was added and incubated for 1 hr at room temperature. Next, 50 μL of a 25 mg/mL slurry of streptavidin-coated SPA beads in PBS with 0.1 M EDTA, pH 8.0 was added. The plate was then sealed and centrifuged for 8 mins at 2000 rpm, and the signal was detected by measuring for 30 sec/well in a Packard TopCount NXT Scintillation Counter. The IC50 values of the inhibition of SGK1 and SGK2 activities by GSK 650394 were calculated from these data using GraphPad Prism 3 Software.

Cell lines

LNCaP cells

Preparation method

The solubility of this compound in DMSO is > 10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 °C for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 °C for several months.

Reacting condition

~ 10 μM; 7 days

Applications

In LNCaP cells, GSK 650394 inhibited androgen-mediated enhancement of Nedd4-2 phosphorylation. In addition, GSK 650394 also significantly inhibited androgen-stimulated growth of LNCaP cells, with an IC50 value of ~ 1 μM.

References:

[1]. Sherk A B, Frigo D E, Schnackenberg C G, et al. Development of a small-molecule serum-and glucocorticoid-regulated kinase-1 antagonist and its evaluation as a prostate cancer therapeutic. Cancer research, 2008, 68(18): 7475-7483.