Ipatasertib (GDC-0068) is a selective, ATP-competitive pan-Akt inhibitor that inhibits Akt1 (IC50:5 nM), Akt2 (IC50:18 nM), and Akt3 (IC50:8 nM). Ipatasertib (GDC-0068) can lead to p53-independent PUMA activation by inhibiting Akt, thereby activating FoxO3a and NF-κB simultaneously, directly binding to the PUMA promoter, upregulating PUMA transcription and Bax-mediated intrinsic mitochondrial apoptosis.

Akt2:18 nM, Akt1:5 nM, Akt3:8 nM

方法：在 Ipatasertib (GDC-0068) （1-20 μM） 处理 HCT116 细胞后的 0、3、6、12 和 24 小时，通过 CCK-8 在 HCT116 中检测细胞活力，研究 ipatasertib 如何影响肿瘤进展。
结果：HCT116 细胞活力随着剂量或时间的增加而显着下降， Ipatasertib (GDC-0068) 可以以剂量和时间依赖性方式抑制细胞增殖。[1]
方法： Ipatasertib (GDC-0068) （10μM）处理HCT116细胞，通过蛋白质印迹分析 HCT116 WT 和 p53−/− 中 p53 或 PUMA 的表达；通过实时 qPCR 分析 WT、p53−/−HCT116 和 DLD1 中 Ipatasertib (GDC-0068) 诱导的 PUMA mRNA，并将其标准化为管家基因 β-actin。
结果：Ipatasertib (GDC-0068) 处理随着剂量的增加，PUMA的表达水平逐渐上调；在 WT （HCT116， RKO）、p53 突变体 （DLD1， HT29） 和 p53 中均观察到这种上调；Ipatasertib (GDC-0068) 可导致 PUMA 的 p53 非依赖性转录激活并抑制细胞增殖。[1]

方法：裸鼠皮下注射HCT116 WT 或 PUMA−/−，模型小鼠服用Ipatasertib (GDC-0068) （30mg / kg，口服，15天）， 计算实验结束时的代表性肿瘤、肿瘤重量和治疗后指定时间点的 c 肿瘤体积，研究PUMA 介导的细胞凋亡对于 ipatasertib 的抗肿瘤活性是否是必需的。
结果：Ipatasertib (GDC-0068) 显着抑制了 WT 肿瘤的生长；免疫组化染色显示，WT和PUMA中P-Akt的表达均降低；Ki67在WT肿瘤中明显降低，但在PUMA中无较大变化；C-Caspase3在WT肿瘤中明显增加，在PUMA中略有增加；Ipatasertib (GDC-0068) 在结肠癌中具有 PUMA 依赖性的抗肿瘤作用。[1]

Kinase Assay: The fluorescence polarization assay for ATP competitive inhibition is done as follows: mPI3Kα dilution solution (90 nM) is prepared in fresh assay buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 5 mM DTT, 0.05% CHAPS) and kept on ice. The enzyme reaction contains 0.5 nM mouse PI3Kα (p110α/p85α complex purified from insect cells), 30 μM PIP2, PF-04691502 (0, 1, 4, and 8 nM), 5 mM MgCl2, and 2-fold serial dilutions of ATP (0–800 μM). Final dimethyl sulfoxide is 2.5%. The reaction is initiated by the addition of ATP and terminated after 30 minutes with 10 mM EDTA. In a detection plate, 15 uL of detector/probe mixture containing 480 nM GST-Grp1PH domain and 12 nM TAMRA tagged fluorescent PIP3 in assay buffer is mixed with 15 uL of kinase reaction mixture. The plate is shaken for 3 minutes, and incubated for 35 to 40 minutes before reading on an LJL Analyst HT.

GDC-0068 is prepared in DMSO and stored, and then diluted with appropriate medium before use[2]. The 384-well plates are seeded with 2,000 cells per well in a volume of 54 μL per well followed by incubation at 37°C under 5% CO2 overnight (~16 hours). Compounds (e.g., GDC-0068) are diluted in DMSO to generate the desired stock concentrations then added in a volume of 6 μL per well. All treatments are tested in quadruplicates. After 4 days incubation, relative numbers of viable cells are estimated using CellTiter-Glo and total luminescence is measured on a Wallac Multilabel Reader. The concentration of drug resulting in IC50 is calculated from a 4-parameter curve analysis (XLfit) and is determined from a minimum of 3 experiments. For cell lines that failed to achieve an IC50, the highest concentration tested (10 μM) is listed[2].