Yaguchi Shin-ichi, Fukui Yasuhisa, Koshimizu Ichiro, Yoshimi Hisashi, Matsuno Toshiyuki, Gouda Hiroaki, Hirono Shuichi, Yamazaki Kanami, Yamori Takao
Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Ariake, Tokyo, Japan.
J Natl Cancer Inst. 2006 Apr 19;98(8):545-56. doi: 10.1093/jnci/djj133.
We previously synthesized a novel s-triazine derivative, ZSTK474 [2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine], that strongly inhibited the growth of tumor cells. We identified its molecular target, investigated its effects on cellular signaling pathways, and examined its antitumor efficacy and toxicity in vivo.
We used COMPARE analysis of chemosensitivity measurements from 39 human cancer cell lines and identified phosphatidylinositol 3-kinase (PI3K) as a molecular target for ZSTK474. PI3K was immunoprecipitated from A549 cell lysates, and its activity was measured by assessing the incorporation of 32P into phosphatidylinositol. We used the crystal structure of the PI3K-LY294002 complex to model the binding of ZSTK474 to PI3K (where LY294002 is a known PI3K inhibitor). PI3K downstream activity was analyzed by immunoblotting. Antitumor activity of ZSTK474 was examined against A549, PC-3, and WiDr xenografts in nude mice. Phosphorylation of Akt, a serine/threonine protein kinase and a major signaling component downstream of PI3K, was assessed in vivo by immunohistochemistry.
PI3K was identified as a molecular target for ZSTK474 by COMPARE analysis. We confirmed that ZSTK474 directly inhibited PI3K activity more efficiently than the PI3K inhibitor LY294002. At concentrations of 1 microM, ZSTK474 and LY2194002 reduced PI3K activity to 4.7% (95% confidence interval [CI] = 3.2% to 6.1%) and 44.6% (95% CI = 38.9% to 50.3%), respectively, of the untreated control level. Molecular modeling of the PI3K-ZSTK474 complex indicated that ZSTK474 could bind to the ATP-binding pocket of PI3K. ZSTK474 inhibited phosphorylation of signaling components downstream from PI3K, such as Akt and glycogen synthase kinase 3beta, and mediated a decrease in cyclin D1 levels. ZSTK474 administered orally to mice had strong antitumor activity against human cancer xenografts without toxic effects in critical organs. Akt phosphorylation was reduced in xenograft tumors after oral administration of ZSTK474.
ZSTK474 is a new PI3K inhibitor with strong antitumor activity against human cancer xenografts without toxic effects in critical organs. ZSTK474 merits further investigation as an anticancer drug.
我们之前合成了一种新型的 s - 三嗪衍生物 ZSTK474 [2 - (2 - 二氟甲基苯并咪唑 - 1 - 基) - 4,6 - 二吗啉基 - 1,3,5 - 三嗪]，它能强烈抑制肿瘤细胞的生长。我们确定了其分子靶点，研究了它对细胞信号通路的影响，并在体内检测了其抗肿瘤功效和毒性。
我们对来自 39 种人类癌细胞系的化学敏感性测量结果进行了 COMPARE 分析，并确定磷脂酰肌醇 3 - 激酶（PI3K）是 ZSTK474 的分子靶点。从 A549 细胞裂解物中免疫沉淀 PI3K，并通过评估 32P 掺入磷脂酰肌醇的情况来测量其活性。我们利用 PI3K - LY294002 复合物的晶体结构来模拟 ZSTK474 与 PI3K 的结合（其中 LY294002 是一种已知的 PI3K 抑制剂）。通过免疫印迹分析 PI3K 的下游活性。在裸鼠中检测 ZSTK474 对 A549、PC - 3 和 WiDr 异种移植瘤的抗肿瘤活性。通过免疫组织化学在体内评估 Akt（一种丝氨酸/苏氨酸蛋白激酶，是 PI3K 下游的主要信号成分）的磷酸化情况。
通过 COMPARE 分析确定 PI3K 是 ZSTK474 的分子靶点。我们证实 ZSTK474 比 PI3K 抑制剂 LY294002 更有效地直接抑制 PI3K 活性。在 1 microM 的浓度下，ZSTK474 和 LY2194002 分别将 PI3K 活性降低至未处理对照水平的 4.7%（95%置信区间[CI]=3.2%至 6.1%）和 44.6%（95%CI = 38.9%至 50.3%）。PI3K - ZSTK474 复合物的分子模拟表明 ZSTK474 可以结合到 PI3K 的 ATP 结合口袋。ZSTK474 抑制 PI3K 下游信号成分的磷酸化，如 Akt 和糖原合酶激酶 3β，并介导细胞周期蛋白 D1 水平的降低。给小鼠口服 ZSTK474 对人类癌症异种移植瘤具有强大的抗肿瘤活性，且对关键器官无毒性作用。口服 ZSTK474 后，异种移植瘤中的 Akt 磷酸化水平降低。
ZSTK474 是一种新型的 PI3K 抑制剂，对人类癌症异种移植瘤具有强大的抗肿瘤活性，且对关键器官无毒性作用。ZSTK474 作为一种抗癌药物值得进一步研究。
