AT7867 is a potent ATP-competitive inhibitor of Akt1/2/3 and p70S6K/PKA with IC50 of 32 nM/17 nM/47 nM and 85 nM/20 nM, respectively; little activity outside the AGC kinase family.

Akt3:47 nM, PKA:20 nM, p70 S6K:85 nM, Akt2:17 nM, Akt1:32 nM

AT7867不仅抑制与AGC激酶结构相关的p70S6K和PKA，其IC50分别为20 nM和85 nM，而且对Akt2显示出Ki为18 nM的ATP竞争性活性。在带有PTEN或PIK3CA突变的细胞系中，AT7867展现出抗增殖效果，并且在MES-SA、MDA-MB-468、MCF-7、HCT116和HT29细胞系中显示出极大的潜力，其IC50分别为0.94 μM、2.26 μM、1.86 μM、1.76 μM和3.04 μM。AT7867还能抑制U87 mg、PC-3和DU145细胞的生长，其IC50分别为8.22 μM、10.37 μM和11.86 μM。通过抑制GSK-3β的磷酸化，AT7867抑制人类肿瘤细胞中的Akt活性，IC50为2-4 μM。此外，AT7867还诱导U87 mg细胞中Akt直接底物包括促凋亡转录因子FKHR (FoxO1a)、FKHRL1 (FoxO3a)以及下游目标S6RP的磷酸化。[1]

AT7867在小鼠中经口服途径展现了44%的生物利用度。通过20 mg/kg的腹腔注射或90 mg/kg的口服给药，AT7867能够在MES-SA异种移植瘤中增加裂解的PARP水平。在MES-SA异种移植瘤或U87 mg异种移植瘤中，AT7867显著抑制了肿瘤的生长，其T/C分别为0.37和0.51。[1]

In vitro kinase assays : Kinase assays for Akt2, PKA, p70S6K, and CDK2/cyclin A are all carried out in a radiometric filter binding format. Assay reactions are set up in the presence of AT7867. For Akt2, the Akt2 enzyme and 25 μM Aktide-2T peptide (HARKRERTYSFGHHA) are incubated in 20 mM MOPS (pH 7.2), 25 mM β-glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 10 μg/mL bovine serum albumin, and 30 μM ATP (1.16 Ci/mmol) for 4 hours. For PKA, the PKA enzyme and 50 μMpeptide (GRTGRRNSI) are incubated in 2 mM MOPS (pH 7.2), 25 mM β-glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM orthovanadate, 1 mM DTT, and 40 μM ATP (0.88 Ci/mmol) for 20 minutes. For p70S6K, the p70S6K enzyme and 25 μMpeptide substrate (AKRRRLSSLRA) are incubated in 10 mM MOPS (pH 7), 0.2 mM EDTA, 1 mM MgCl2, 0.01% β-mercaptoethanol, 0.1 mg/mL bovine serum albumin, 0.001% Brij-35, 0.5% glycerol, and 15 μM ATP (2.3 Ci/mmol) for 60 min. For CDK2, the CDK2/cyclin A enzyme and 0.12 μg/mL histone H1 are incubated in 20 mM MOPS (pH 7.2), 25 mM β-glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 0.1 mg/mL bovine serum albumin, and 45 μM ATP (0.78 Ci/mmol) for 4 hours. Assay reactions are stopped by adding an excess of orthophosphoric acid, and the stopped reaction mixture is then transferred to Millipore MAPH filter plates and filtered. The plates are then washed, scintillant is added, and radioactivity is measured by scintillation counting on a Packard TopCount. IC50 values are calculated from replicate curves using GraphPad Prism software. Akt1 and Akt3 enzyme assays are carried out.

Cells are plated in 96-well microplates at 5 × 103 per well in medium supplemented with 10% fetal bovine serum and grown for 24 hours before treatment with AT7867. AT7867 or vehicle control is added to the cells for 72 hours. Following this, Alamar Blue solution is added. The IC50 value for AT7868 is calculated in GraphPad Prism using nonlinear regression analysis and a sigmoidal dose-response (variable slope) equation.(Only for Reference)