Abstract

Protein kinase B/AKT kinase is the core component of the phosphatidylinositol 3-kinase/AKT signaling pathway, which is frequently hyperactivated in human cancers. We designed and synthesized a series of 2-pyrimidyl-5-amidothiazole compounds based on the ATP binding site of AKT, and the most potent compound, (S)-N-(1-amino-3-(2,4-dichlorophenyl)propan-2-yl)-2-(2-(methylamino)pyrimidin-4-yl)thiazole-5-carboxamide (DC120), was identified to inhibit AKT activity in vitro with an EC₅₀ of 153 nM by a fluorescence resonance energy transfer-based Z′-LYTE assay. The antitumor effect of DC120 was tested on human CNE2 and MDA-MB-453 cell lines and the CNE2 xenograft model. The results showed that DC120 could obviously inhibit the proliferation of CNE2 and MDA-MB-453 cells via induction of apoptosis, with the evidence of increases in sub-G₁ and annexin V-positive cells, characteristic morphologic changes of apoptosis in the nucleus, and cleaved caspase-3. Further study showed that MDA-MB-453 cells transfected with constitutively activated AKT1 were more sensitive to DC120,whereas CNE2 cells with knockdown of AKT1 expression by short hairpin RNA were more resistant to DC120. Of more importance, DC120 partially attenuated the phosphorylation levels of forkhead transcription factor (FKHR), FKHRL1, glycogen synthase kinase 3β, and mammalian target of rapamycin in a dose-dependent and time-dependent fashion and led to an increase in the nuclear accumulation of exogenous FKHR in cancer cells. In addition, DC120 at 20 mg/kg/day inhibited the CNE2 xenograft tumor growth with a treated group/control group ratio of 38.1%, accompanied by increasing terminal deoxynucleotidyl transferasedUTP nick-end labeling-positive cells in the tumor sample. In addition, DC120 induced a feedback loop to activate the mitogen-activated protein kinase pathway and treatment with mitogen-activated protein kinase kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and DC120 synergistically induced cancer cell apoptosis. These data provide validation for the development of DC120 to treat cancers displaying elevated levels of AKT.