Uprosertib (GSK2141795) is a potent, selective Akt broad-spectrum inhibitor with IC50 values of 180/328/38 nM for Akt1/Akt2/Akt3.

Akt1:180 nM, Akt2:328 nM, Akt3:38 nM

方法：K562、COLO205、SKNBE2和OVCAR8细胞混合物的裂解物用0 、2.5 nM、25 nM、250 nM、2.5 μM或25 μM的游离化合物Uprosertib (GSK2141795)在4°C的端对端摇床上预孵育45分钟。随后，裂解物与珠(偶联Akt探针或kinobads)在4°C下孵育1小时，进行定性和定量实验。
结果：Uprosertib抑制kinobead结合，Akt1的IC50值为180 nM, Akt2的IC50值为328 nM, Akt3的IC50值为38 nM；Akt1的KD值为16 nM, Akt2的KD值为49 nM, Akt3的KD值为5 nM 。[1]
方法：用 10 μM Uprosertib处理 HAP1、HAP1 RNF43 KO 和 HAP1 PWWP2B KO 细胞 48 小时，并使用 MTS 测定法评估Uprosertib的抑制作用，并检测对细胞系活力的影响。
结果：Uprosertib以剂量依赖性方式降低细胞活力。[2]
方法：在存在或不存在乳酸（0-20 mM）的情况下，用uprosertib（1μM至15μM）处理HCT116和LS174T细胞系72小时，并使用SRB测定法测定生物量；在存在或不存在乳酸（10或20mM）的情况下，用uprosertib（5或10μM）处理细胞24小时，并使用Caspase-Glo 3/7测定测量细胞凋亡
结果：Uprosertib在两种细胞系中诱导剂量依赖性细胞毒性；uprosertib诱导HCT116和LS174T细胞系中caspase-3/7激活的剂量依赖性增加。[3]

方法：用Uprosertib (GSK2141795)（10mg/kg/day，口服）来研究对 MKN45 异种移植模型小鼠生长的体内疗效。
结果：uprosertib 可在 3 周时显着抑制肿瘤生长，在 KMN45 异种移植模型小鼠中的抑制率为27%。[2]

Selectivity profiling experiments: The lysates (5 mg of total protein each) are preincubated with 0 (DMSO control), 2.5 nM, 25 nM, 250 nM, 2.5 μM or 25 μM free compound (GSK690693 or GSK2141795) on an end-over-end shaker for 45 min at 4 °C. Subsequently, lysates are incubated with beads (coupled Akt probe or kinobeads) for 1 h at 4 °C, for both qualitative and quantitative experiments. The beads are washed with 1× CP buffer and collected by centrifugation. Bound proteins are eluted with 2× NuPAGE LDS sample buffer, and eluates are reduced and alkylated by 50 mM dithiothreitol and 55 mM iodoacetamide.

Cell lines are typically grown in RPMI 160 medium containing 10% FBS. Some cell lines are grown in media specified by the vendor. A 3-day proliferation assay using CellTiter-Glo is performed to measure the growth inhibition by the compounds at 0–30 μM. Cell growth is determined relative to untreated (DMSO) controls. EC50's are calculated from inhibition curves using a 4- or 6-parameter fitting algorithm in the Assay Client application.(Only for Reference)