Reindl Wolfgang, Strebhardt Klaus, Berg Thorsten
Department of Molecular Biology, Max Planck Institute of Biochemistry, Munich Center for Integrated Protein Science (CIPSM), Am Klopferspitz 18, 82152 Martinsried, Germany.
Anal Biochem. 2008 Dec 15;383(2):205-9. doi: 10.1016/j.ab.2008.08.014. Epub 2008 Aug 22.
The serine/threonine kinase polo-like kinase 1 (Plk1) is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its substrates and its intracellular anchoring sites via its polo-box domain (PBD), which is unique to the family of polo-like kinases. Therefore, inhibition of the Plk1 PBD has been suggested as an approach to the inhibition of Plk1 that circumvents specificity problems associated with the inhibition of the conserved adenosine triphosphate (ATP) binding pocket. Here we report on the development of a high-throughput assay based on fluorescence polarization that allows the discovery of small-molecule inhibitors of the Plk1 PBD. The assay is based on binding of the Plk1 PBD to a phosphothreonine-containing peptide comprising its optimal binding motif with a K(d) of 26+/-2 nM. It is stable with regard to dimethyl sulfoxide (DMSO) and time, and it has a Z' value of 0.73+/-0.06 in a 384-well format.
丝氨酸/苏氨酸激酶波罗样激酶1（Plk1）在多个有丝分裂过程中起关键作用，并且已被确立为肿瘤患者的不良预后标志物。Plk1通过其波罗盒结构域（PBD）定位于其底物及其细胞内锚定位点，该结构域是波罗样激酶家族所特有的。因此，有人提出抑制Plk1的PBD是一种抑制Plk1的方法，可避免与抑制保守的三磷酸腺苷（ATP）结合口袋相关的特异性问题。在此，我们报告了一种基于荧光偏振的高通量检测方法的开发，该方法可用于发现Plk1 PBD的小分子抑制剂。该检测基于Plk1 PBD与包含其最佳结合基序的含磷酸苏氨酸肽的结合，解离常数（K(d)）为26±2 nM。它在二甲基亚砜（DMSO）和时间方面很稳定，在384孔板形式下的Z'值为0.73±0.06。
