State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, 651 Dongfeng Road East, Guangzhou, China.
Mol Pharmacol. 2012 Aug;82(2):189-98. doi: 10.1124/mol.111.077271. Epub 2012 May 2.
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.
蛋白激酶 B/AKT 激酶是磷脂酰肌醇 3-激酶/AKT 信号通路的核心组成部分，该通路在人类癌症中经常过度激活。我们基于 AKT 的 ATP 结合位点设计并合成了一系列 2-嘧啶基-5-酰胺噻唑化合物，最有效的化合物（S）-N-（1-氨基-3-（2,4-二氯苯基）丙-2-基）-2-（2-（甲基氨基）嘧啶-4-基）噻唑-5-甲酰胺（DC120），通过基于荧光共振能量转移的 Z'-LYTE 测定法，以 EC（50）为 153 nM 的浓度在体外抑制 AKT 活性。在 CNE2 和 MDA-MB-453 细胞系和 CNE2 异种移植模型中测试了 DC120 的抗肿瘤作用。结果表明，DC120 通过诱导细胞凋亡明显抑制 CNE2 和 MDA-MB-453 细胞的增殖，表现为亚 G1 期和膜联蛋白 V 阳性细胞增加、细胞核凋亡特征性形态改变和 cleaved caspase-3。进一步的研究表明，转染组成性激活 AKT1 的 MDA-MB-453 细胞对 DC120 更敏感，而用短发夹 RNA 敲低 AKT1 表达的 CNE2 细胞对 DC120 更耐药。更重要的是，DC120 以剂量和时间依赖的方式部分降低叉头转录因子（FKHR）、FKHRL1、糖原合酶激酶 3β 和雷帕霉素哺乳动物靶蛋白的磷酸化水平，并导致外源性 FKHR 在癌细胞中的核积累增加。此外，20mg/kg/天的 DC120 抑制 CNE2 异种移植肿瘤生长，处理组/对照组比值为 38.1%，同时肿瘤样本中端末脱氧核苷酸转移酶 UTP 缺口末端标记阳性细胞增加。此外，DC120 诱导反馈环激活丝裂原活化蛋白激酶通路，并用丝裂原活化蛋白激酶激酶抑制剂 1,4-二氨基-2,3-二氰基-1,4-双（甲基硫代）丁二烯（U0126）和 DC120 联合处理可协同诱导癌细胞凋亡。这些数据为开发 DC120 治疗 AKT 水平升高的癌症提供了验证。
