ZSTK474 is an ATP-competitive inhibitor of class I phosphatidylinositol 3 kinase isoforms
ZSTK474 is an ATP-competitive inhibitor of class I phosphatidylinositol 3 kinase isoforms
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DOI：
10.1111/j.1349-7006.2007.00580.x
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发表时间：
2007-10-01
期刊：
影响因子：
5.7
通讯作者：
Yamori, Takao
中科院分区：
文献类型：
--
作者：
Kong, Dexin;Yamori, Takao
I类磷脂酰肌醇3激酶(PI3K)使磷脂酰肌醇4，5-二磷酸磷酸化，生成磷脂酰肌醇3，4，5-三磷酸。这些分子在基本的细胞反应中起着重要作用。已知I类PI3K的四种亚型具有不同的功能，它们活动的异常与癌症和炎症等各种疾病有关。我们之前发现了一种新的PI3K抑制剂ZSTK474，它在体内对人肿瘤移植瘤具有很强的抗肿瘤活性，但没有明显的毒性。然而，其分子作用方式还没有得到详细的研究。我们先前的研究仅表明ZSTK474可能与ATP竞争PI3K伽马的ATP结合口袋。在本研究中，我们使用了体外均一的时间分辨荧光激酶实验来检测ZSTK474是否是PI3K的ATP竞争抑制物，以及ZSTK474的抑制活性是否是异构体特异性的。Lineweaver-Burk图分析表明，ZSTK474以ATP竞争的方式抑制所有四种PI3K亚型。在所有的PI3K亚型中，ZSTK474对PI3K Delta的抑制作用最强，K-I为1.8 nM，其他亚型在较高剂量时受到抑制。我们还使用了激酶活性ELISA法来确定ZSTK474是否抑制了雷帕霉素的哺乳动物靶点，雷帕霉素是一种作用于PI3K下游的关键激酶，可促进蛋白质合成和细胞增殖。即使在100亩M的浓度下，ZSTK474对雷帕霉素活性的哺乳动物靶标的抑制作用也相当弱。这些结果表明，ZSTK474是一种ATP竞争性的泛类PI3K抑制剂。
Class I phosphatidylinositol 3 kinases (PI3K) phosphorylate phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol 3,4,5-trisphosphate. These molecules play an important role in fundamental cellular responses. Four isoforms of class I PI3K are known to have different functions, and abnormalities in their activities have been related to various diseases such as cancer and inflammation. We previously identified a novel PI3K inhibitor, ZSTK474, which showed potent antitumor activity in vivo against a human cancer xenograft without observable toxicity. However, the mode of its molecular action was not investigated in detail. Our previous study only suggested that ZSTK474 possibly competes with ATP for the ATP-binding pocket of PI3K gamma. In the present study, we have used an in vitro homogenous time-resolved fluorescence kinase assay to examine whether ZSTK474 is indeed an ATP-competing inhibitor of PI3K, and also to determine whether the inhibitory activity of ZSTK474 was isoform-specific. Lineweaver-Burk plot analysis revealed that ZSTK474 inhibits all four PI3K isoforms in an ATP-competitive manner. Among all of the PI3K isoforms, PI3K delta was inhibited most potently by ZSTK474 with a K-i of 1.8 nM, and the other isoforms were inhibited at higher doses. We have also used a kinase activity ELISA to determine whether ZSTK474 inhibits mammalian target of rapamycin, a key kinase acting downstream of PI3K to promote protein synthesis and cell proliferation. Even at a concentration of 100 mu M, ZSTK474 inhibited mammalian target of rapamycin activity rather weakly. These results indicate that ZSTK474 is an ATP-competitive pan-class I PI3K inhibitor.