GSK 650394 is an inhibitor of serum- and glucocorticoid-regulated kinases (SGK) that inhibits SGK1 and SGK2 (IC50=62/103 nM). GSK 650394 has antitumor activity and also inhibits osteoclast differentiation and prevents bone loss.

SGK1:62 nM, SGK2:103 nM

METHODS: HB1 cells were treated with Grazoprevir (6-30 nM) and G418 (0.5 mg/mL) for 3 weeks for in vitro resistance selection.
RESULTS: Grazoprevir showed good mutational potency in all resistant variants and inhibited R155 and D168 mutations in particular. [1]
METHODS: 315 compounds with electrophilic moieties were experimentally screened to determine if they would covalently bind to the active site cysteine of SARS-CoV-2 3CLpro.
RESULTS: Among the compounds, Grazoprevir was able to inhibit the activity of SARS-CoV-2 3CLpro. [2]

METHODS: To validate in vivo efficacy against chronic HCV, Grazoprevir (1 mg/kg) was administered orally to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections twice daily for seven days.
RESULTS: Grazoprevir was able to suppress viral load by 4-5 logs in vivo. [1]
METHODS: To assess in vivo pharmacokinetics, Grazoprevir (5 mg/kg) was administered orally to rats.
RESULTS: Plasma exposure to Grazoprevir was favorable, with an AUC of 0.7 µM h/mL. [3]

Scintillation proximity assay (SPA): SGK1 S422D (60–431 aa; 0.275 μg/mL final concentration) or SGK2 (0.875 μg/mL final concentration) are 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 min at 30°C. SGK2 is 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 is prepared in the reaction buffer. In a 96-well plate, 5 μL of GSK650394 is added to 25 μL of the activated enzyme mixture. To this, 20 μL of the CROSStide mixture is added and incubated for 1 h 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 is added. The plate is then sealed and centrifuged for 8 min at 2000 rpm, and the signal is 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 GSK650394 are calculated from these data using GraphPad Prism 3 Software.

LNCaP cells are plated at a density of 5,000 cells per well in 96-well plates in 100 μL PRF-RPMI 1640, supplemented with 8% CS-FBS, 0.1 mM NEAA, and 1 mM NaPyr. At day three, cells are treated with hormone with or without GSK650394 by removing 50 μL of the media and replacing this with 50 μL of PRF-RPMI 1640 with 8% CS-FBS, NEAA, NaPyr containing a 2X concentration of the appropriate hormone/inhibitor treatment. At days 5 and 7, the treatment is repeated. On the tenth day, the media is removed and the relative cell number is measured using the FluoReporter Blue assay according to the manufacturer's instructions.(Only for Reference)