A-674563 2HCl is an Akt1 inhibitor with Ki of 11 nM, modest potent to PKA and >30-fold selective for Akt1 over PKC.

PKA:16 nM (Ki), CDK2:46 nM (Ki), Akt1:11 nM (Ki), GSK-3β:110 nM (Ki)

A-674563 在细胞内降低Akt下游靶点的磷酸化，并在体外减缓肿瘤细胞的增殖（EC50: 0.4 μM）[1]。A563 (0-10 μM) 显著降低STS细胞中GSK3和MDM2的磷酸化。对所有STS细胞系，A563展现出抑制效果，48小时内的IC50值介于0.22 μM至0.35 μM之间。A563在STS细胞中诱发G2期细胞周期阻滞和凋亡。A563 (1 μM/12小时) 不依赖于p53上调GADD45A的表达[2]。在培养的人类黑色素瘤细胞中，A-674563 (10-1000 nM) 具有抗增殖和细胞毒性，通过启动凋亡死亡，并且这种效果可被caspase抑制剂所抑制，通过Akt依赖性和非依赖性机制抑制黑色素瘤细胞[3]。加入到U937和AmL祖细胞中，A-674563显示出细胞毒性和抗增殖作用，激活caspase-3/9并在U937及AmL祖细胞中诱发凋亡，同时在AmL细胞中调节其他信号传导，阻断Akt。

在PC-3前列腺癌异种移植模型中，A-674563（40 mg/kg/d，口服）未表现出显著的单药疗效，但联合疗法（A-674563+紫杉醇）的疗效显著提升。在口服葡萄糖耐量测试中，A-674563（20, 100 mg/kg）能增加血浆胰岛素水平[1]。A563（20 mg/kg/bid；口服）显示出缓慢的肿瘤生长速率和在肿瘤体积上的显著差异，同时未引起小鼠明显的体重损失。A563处理的肿瘤表达增加的GADD45α水平和减少的PCNA（细胞核增殖标志）水平。此外，A563处理样本中TUNEL染色水平（凋亡标志）有所增加[2]。A-674563（25, 100 mg/kg，每日灌肠）有效抑制了小鼠A375异种移植瘤的生长[3]。A-674563（15, 40 mg/kg）注射抑制了U937异种移植瘤体内的生长，并改善了小鼠的生存率。

The cells on 96-well plates are gently washed with 200 μL of PBS. Alamar Blue reagent is diluted 1:10 in normal growth media. The diluted Alamar Blue reagent (100 μL) is added to each well on the 96-well plates and incubated until the reaction is complete. The analysis is done using a fmax Fluorescence Microplate Reader, set at the excitation wavelength of 544 nm and the emission wavelength of 595 nm. Data are analyzed using SOFTmax PRO software.

A-674563 is formulated in 5% dextrose.Immunocompromised male SCID mice are at 6 to 8 weeks of age. The 1×106 3T3-Akt1 or 2×106 MiaPaCa-2 and PC-3 cells in 50% Matrigel are inoculated s.c. into the flank. For early treatment studies, mice are randomly assigned to treatment groups and therapy is initiated the day after inoculation. Ten animals are assigned to each group, including controls. For established tumor studies, tumors are allowed to reach a designated size and mice are assigned to treatment groups of equal tumor size (n=10 mice per group). Tumor size is evaluated by twice-weekly measurements with digital calipers. Tumor volume is estimated using the formula: V=L×W2/2. A-443654 is given s.c. in a vehicle of 0.2% HPMC. A-674563 is given orally in a vehicle of 5% dextrose. Gemcitabine and paclitaxel are added to the assay.