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

Akt1:11 nM(Ki)

A-674563是通过将A-443654中的吲哚基团替换为苯基团并获得口服活性而得到的。A-674563能够以0.4μM的EC50抑制肿瘤细胞的增殖。[1] A-674563本身不抑制Akt的磷酸化，但能以剂量依赖性方式阻断Akt下游靶点的磷酸化。A-674563引起的Akt阻断导致STS细胞下游靶点磷酸化减少以及肿瘤细胞生长抑制。A-674563在STS细胞中诱导G2细胞周期阻滞及凋亡。[2]

在口服葡萄糖耐量测试中，20 mg/kg A-674563可提高血浆胰岛素水平。A-674563单药治疗未显示出明显的抑瘤活性；与紫杉醇单药治疗相比，联合治疗的疗效显著提高。[1]治疗的小鼠（20 mg/kg/bid，p.o.）展现出比对照组更慢的肿瘤生长速率，并在研究终点时肿瘤体积减少了50%以上。[2]在小鼠中，A-674563的口服生物利用度为67%，其药代动力学（PK）特性显著提高，但其活性比A-443654低70倍。[3]

Akt Kinase Assay: The kinase assay uses His-Akt1 and a biotinylated mouse Bad peptide as substrate. The kinase assay is carried out at room temperature for 30 minutes in 50 μL of reaction buffer [20 mM HEPES, pH 7.5, 10 mM MgCl2, 0.1% (w/v) Triton X-100, 5 μM ATP (Km = 40 μM), 5 μM peptide (Km = 15 μM), 1 mM DTT, 60 ng of Akt1, and 0.5 μCi of [γ-33P]ATP] in the presence of different concentrations of A-674563. Each reaction is stopped by adding 50 μL of termination buffer (0.1 M EDTA, pH 8.0, and 4 M NaCl). The biotinylated Bad peptides are immobilized on streptavidin-coated FLASH plates. After being washed with PBS-Tween 20 (0.05%), the 33P phosphopeptide captured on the FLASH plates is measured with a TopCount Packard Instruments γ counter.

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 M) is added to each well on the 96-well plates and incubated until the reaction is complete as per manufacturer's instructions. Analysis is done using an fmax Fluorescence Microplate Reader, set at the excitation wavelength of 544 nm and emission wavelength of 595 nm. Data are analyzed using SOFTmax PRO software provided by the manufacturer. (Only for Reference)