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

方法：LNCaP 细胞用 GSK 650394 (0.1-10 µM) 和 R1881 (1 nM) 处理 24-48 h，通过 Western Blot 检测靶点蛋白表达水平。
结果：GSK 650394 抑制了雄激素介导的 Nedd4-2 磷酸化增强，表明 GSK 650394 拮抗了细胞中的 SGK1 活性。[1]
方法：肿瘤细胞 HeLa 和 HepG2 用 GSK 650394 (3.12-12.5 µM) 处理 24-72 h，通过 MTT assay 测定细胞活力。
结果：用 GSK 650394 处理的细胞以剂量依赖的方式显示出显著的生长抑制作用。[2]

方法：为研究对骨质疏松症的作用，将 GSK 650394 (10-30 mg/kg) 腹腔注射给卵巢切除诱导的骨丢失小鼠，每周三次，持续八周。
结果：GSK 650394 治疗改善了卵巢切除诱导的骨丢失小鼠的骨密度。GSK 650394 治疗可以提高体内抗氧化酶的能力。研究表明，GSK 650394 可以抑制破骨细胞的活化，因此可能是治疗破骨细胞活化相关骨质疏松症的潜在治疗试剂。[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)