Knockaert Marie, Lenormand Philippe, Gray Nathanael, Schultz Peter, Pouysségur Jacques, Meijer Laurent
Station Biologique de Roscoff, CNRS, B.P.74, 29682 Roscoff Cedex, Bretagne, France.
Oncogene. 2002 Sep 19;21(42):6413-24. doi: 10.1038/sj.onc.1205908.
Chemical inhibitors of cyclin-dependent kinases (CDKs) have a great therapeutic potential against various proliferative and neurodegenerative disorders. Intensive screening of a combinatorial chemistry library of 2,6,9-trisubstituted purines has led to the identification of purvalanol, one of the most potent and selective CDK inhibitors to date. In preliminary studies, this compound demonstrates definite anti-mitotic properties, consistent with its nanomolar range efficiency towards purified CDK1 and CDK2. However, the actual intracellular targets of purvalanol remain to be identified, and a method for the determination of its in vivo selectivity was developed. In this technique, cell extracts were screened for purvalanol-interacting proteins by affinity chromatography on immobilized inhibitor. In addition to CDK1, p42/p44 MAPK were found to be two major purvalanol-interacting proteins in five different mammalian cell lines (CCL39, PC12, HBL100, MCF-7 and Jurkat cells), suggesting the generality of the purvalanol/p42/p44 MAPK interaction. The Chinese hamster lung fibroblast cell line CCL39 was used as a model to investigate the anti-proliferative properties of purvalanol. The compound inhibited cell growth with a GI(50) value of 2.5 microM and induced a G2/M block when added to exponentially growing cells. It did not appear to trigger massive activation of caspase. We next tested whether CDKs and p42/p44 MAPK were actually targeted by the compound in vivo. p42/p44 MAPK activity was visualized using an Elk-Gal4 luciferase reporter system and CDK1 activity was detected by the phosphonucleolin level. When cells were treated with purvalanol, p42/p44 MAPK and CDK1 activities were inhibited in a dose-dependent manner. Furthermore, purvalanol inhibited the nuclear accumulation of p42/p44 MAPK, an event dependent on the catalytic activity of these kinases. We conclude that the anti-proliferative properties of purvalanol are mediated by inhibition of both p42/p44 MAPK and CDKs. These observations highlight the potency of moderate selectivity compounds and encourage the search for new therapeutics which simultaneously target distinct but relevant pathways of cell proliferation.
细胞周期蛋白依赖性激酶（CDK）的化学抑制剂在治疗各种增殖性和神经退行性疾病方面具有巨大的治疗潜力。对2,6,9-三取代嘌呤的组合化学文库进行深入筛选，已鉴定出嘌呤醇，它是迄今为止最有效和最具选择性的CDK抑制剂之一。在初步研究中，该化合物表现出明确的抗有丝分裂特性，与其对纯化的CDK1和CDK2的纳摩尔级效率一致。然而，嘌呤醇的实际细胞内靶点仍有待确定，并且开发了一种测定其体内选择性的方法。在这项技术中，通过固定化抑制剂的亲和色谱法筛选细胞提取物中与嘌呤醇相互作用的蛋白质。除了CDK1外，在五种不同的哺乳动物细胞系（CCL39、PC12、HBL100、MCF-7和Jurkat细胞）中发现p42/p44 MAPK是两种主要的与嘌呤醇相互作用的蛋白质，这表明嘌呤醇/p42/p44 MAPK相互作用具有普遍性。中国仓鼠肺成纤维细胞系CCL39被用作研究嘌呤醇抗增殖特性的模型。该化合物抑制细胞生长，GI(50)值为2.5 microM，当添加到指数生长的细胞中时诱导G2/M期阻滞。它似乎没有引发半胱天冬酶的大量激活。接下来，我们测试了CDK和p42/p44 MAPK在体内是否真的是该化合物的靶点。使用Elk-Gal4荧光素酶报告系统观察p42/p44 MAPK活性，并通过磷酸核仁素水平检测CDK1活性。当细胞用嘌呤醇处理时，p42/p44 MAPK和CDK1活性以剂量依赖性方式受到抑制。此外，嘌呤醇抑制p42/p44 MAPK的核积累，这一事件依赖于这些激酶的催化活性。我们得出结论，嘌呤醇的抗增殖特性是通过抑制p42/p44 MAPK和CDK来介导的。这些观察结果突出了中等选择性化合物的效力，并鼓励寻找同时靶向细胞增殖不同但相关途径的新疗法。
