Echalier Aude, Cot Emilie, Camasses Alain, Hodimont Elsie, Hoh François, Jay Philippe, Sheinerman Felix, Krasinska Liliana, Fisher Daniel
Université Montpellier I, 34000 Montpellier, France.
Chem Biol. 2012 Aug 24;19(8):1028-40. doi: 10.1016/j.chembiol.2012.06.015.
Cdk2 promotes DNA replication and is a promising cancer therapeutic target, but its functions appear redundant with Cdk1, an essential Cdk affected by most Cdk2 inhibitors. Here, we present an integrated multidisciplinary approach to address Cdk redundancy. Mathematical modeling of enzymology data predicted conditions allowing selective chemical Cdk2 inhibition. Together with experiments in Xenopus egg extracts, this supports a rate-limiting role for Cdk2 in DNA replication. To confirm this we designed inhibitor-resistant (ir)-Cdk2 mutants using a novel bioinformatics approach. Bypassing inhibition with ir-Cdk2 or with Cdk1 shows that Cdk2 is rate-limiting for replication in this system because Cdk1 is insufficiently active. Additionally, crystal structures and kinetics reveal alternative binding modes of Cdk1-selective and Cdk2-selective inhibitors and mechanisms of Cdk2 inhibitor resistance. Our approach thus provides insight into structure, functions, and biochemistry of a cyclin-dependent kinase.
细胞周期蛋白依赖性激酶2（Cdk2）促进DNA复制，是一个很有前景的癌症治疗靶点，但其功能似乎与细胞周期蛋白依赖性激酶1（Cdk1）冗余，Cdk1是一种受大多数Cdk2抑制剂影响的必需细胞周期蛋白依赖性激酶。在此，我们提出一种综合多学科方法来解决Cdk冗余问题。酶学数据的数学建模预测了允许选择性化学抑制Cdk2的条件。结合非洲爪蟾卵提取物实验，这支持了Cdk2在DNA复制中的限速作用。为证实这一点，我们使用一种新颖的生物信息学方法设计了抗抑制剂（ir）-Cdk2突变体。用ir-Cdk2或Cdk1绕过抑制作用表明，在该系统中Cdk2对复制具有限速作用，因为Cdk1活性不足。此外，晶体结构和动力学揭示了Cdk1选择性抑制剂和Cdk2选择性抑制剂的替代结合模式以及Cdk2抑制剂抗性机制。因此，我们的方法为细胞周期蛋白依赖性激酶的结构、功能和生物化学提供了深入了解。
