CNRS USR 3151, Protein Phosphorylation and Disease Laboratory, Protein-Protein Interaction Inhibition P2I2 Group, Station Biologique, Roscoff, Bretagne, France.
Biotechnol J. 2011 Jul;6(7):860-70. doi: 10.1002/biot.201100138. Epub 2011 Jun 16.
The protein kinase CDK5 (cyclin-dependent kinase 5) is activated through its association with a cyclin-like protein p35 or p39. In pathological conditions (such as Alzheimer's disease and various other neuropathies), truncation of p35 leads to the appearance of the p25 protein. The interaction of p25 with CDK5 up-regulates the kinase activity and modifies the substrate specificity. ATP-mimetic inhibitors of CDK5 have already been developed. However, the lack of selectivity of such inhibitors is often a matter of concern. An alternative approach can be used to identify highly specific inhibitors that disrupt protein interactions involving protein kinases. We have developed a bioluminescence resonance energy transfer (BRET)-based screening assay in yeast to discover protein-protein interaction inhibitors (P2I2). Here, we present the first use of BRET in yeast for the screening of small molecule libraries. This screening campaign led to the discovery of one molecule that prevents the interaction between CDK5 and p25, thus inhibiting the protein kinase activity. This molecule may give rise to high-specificity drug candidates.
蛋白激酶 CDK5（细胞周期蛋白依赖性激酶 5）通过与细胞周期蛋白样蛋白 p35 或 p39 结合而被激活。在病理条件下（如阿尔茨海默病和各种其他神经病变），p35 的截断导致 p25 蛋白的出现。p25 与 CDK5 的相互作用上调激酶活性并改变底物特异性。已经开发了 CDK5 的 ATP 模拟抑制剂。然而，此类抑制剂缺乏选择性通常令人关注。另一种方法可用于鉴定涉及蛋白激酶的蛋白相互作用的高度特异性抑制剂。我们已经开发了一种基于生物发光共振能量转移（BRET）的酵母筛选测定法来发现蛋白-蛋白相互作用抑制剂（P2I2）。在这里，我们首次在酵母中使用 BRET 进行小分子文库的筛选。该筛选活动发现了一种可防止 CDK5 与 p25 相互作用的分子，从而抑制蛋白激酶活性。这种分子可能会产生高特异性的药物候选物。
