A-674563 is an orally available, ATP-competitive, and reversible inhibitor of Akt (Ki: 11 nM for Akt1) [1]. It exhibits inhibitory activity against PKA and Cdk2 (IC50: 16/46 nM) but is 10- to >1, 800-fold selective for Akt1 versus additional kinases in the CMGC, CAMK, and TK families [1].

GSK-3β:110 nM (Ki), Akt1:11 nM (Ki), CDK2:46 nM (Ki), PKA:16 nM (Ki), ERK2:260 nM (Ki), PKCδ:360 nM (Ki)

A-674563 降低细胞内Akt下游靶点的磷酸化水平，减缓体外肿瘤细胞的增殖 (EC50: 0.4 μM) [1]。A563 (0-10 μM) 显著降低STS细胞中GSK3与MDM2的磷酸化水平。A563 对所有STS细胞系均显示出抑制作用，48小时内IC50值介于0.22 μM至0.35 μM之间。A563 在STS细胞中诱导G2期细胞周期阻滞及凋亡。A563 (1 μM/12 hr) 无论p53是否存在均可上调GADD45A的表达量[2]。在培养的人类黑色素瘤细胞中，A-674563 (10-1000 nM) 具有抗增殖与细胞毒性作用，能诱导黑色素瘤细胞的凋亡死亡，此作用被caspase抑制剂所抑制，且通过Akt依赖及独立机制抑制黑色素瘤细胞[3]。在向U937与AmL前体细胞添加A-674563后，其表现出细胞性毒性与抗增殖作用，能激活caspase-3/9并在U937与AmL前体细胞中诱导凋亡，同时在阻断Akt的同时影响AmL细胞中的其他信号传递[4]。

在PC-3前列腺癌异种移植模型中，A-674563 (40 mg/kg/d, p.o.) 单独治疗没有明显的抗癌活性，但与紫杉醇联合治疗（A-674563+paclitaxel）的疗效显著提高。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; p.o.)表现出缓慢的肿瘤生长速度及显著的肿瘤体积差异，同时不会导致小鼠显著体重减轻。A563处理的肿瘤表现出GADD45α水平增加以及PCNA（一种增殖的核标记物）水平降低。此外，TUNEL检测染色水平（凋亡标记物）在A563处理的样本中增加[2]。A-674563 (25, 100 mg/kg, lavage daily)有效抑制小鼠A375异种移植物生长[3]。A-674563 (15, 40 mg/kg)注射能够抑制U937异种移植物的体内生长，并提高小鼠的生存率[4]。

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.