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 EC(50) 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-G(1) 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.