A subnanomolar fluorescent probe for protein kinase CK2 interaction studies
A subnanomolar fluorescent probe for protein kinase CK2 interaction studies
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DOI：
10.1039/c2ob26022k
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发表时间：
2012-01-01
影响因子：
3.2
通讯作者：
Uri, Asko
中科院分区：
文献类型：
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作者：
Enkvist, Erki;Viht, Kaido;Uri, Asko
嗜酸蛋白激酶CK2的上调已在几种类型的癌症中得到证实。这种认知使得CK2成为癌症化疗药物开发的重要靶点。利用高亲和力和选择性结合到CK2活性位点的小分子荧光探针，可以促进潜在候选药物的表征，确定CK2的结构和澄清功能。我们使用了一种双底物方法来开发一种高效的CK2抑制剂。4,5,6,7-四溴- 1h -苯并咪唑通过不同的连接体与含有多个天冬氨酸残基的肽偶联。抑制剂的设计是通过对抑制剂与酶的催化亚基（CK2 α）的络合物的晶体学分析。通过薄层色谱法测定荧光标记肽5-TAMRA-RADDSDDDDD的磷酸化速率，建立了合成化合物的抑制效力。最有效的抑制剂ARC-1502 （K-i = 0.5 nM）在140个蛋白激酶中显示出对CK2 α的高选择性。用PromoFluor-647标记ARC-1502，使荧光探针ARC-1504对CK2 α和全酶都具有亚纳摩尔亲和力。该探针用于基于荧光各向异性的结合实验，以测量CK2 α的浓度，并表征与CK2 α活性位点结合的非标记配体。
Up-regulation of an acidophilic protein kinase, CK2, has been established in several types of cancer. This cognition has made CK2 an important target for drug development for cancer chemotherapy. The characterization of potential drug candidates, determination of the structure and clarification of the functions of CK2 could be facilitated by the application of small-molecule fluorescent probes that bind to the active site of the enzyme with high affinity and selectivity. We have used a bisubstrate approach for the development of a highly potent inhibitor of CK2. 4,5,6,7-Tetrabromo-1H-benzimidazole was conjugated with peptides containing multiple aspartate residues via different linkers. The design of the inhibitors was by crystallographic analysis of the complex of an inhibitor with the catalytic subunit of the enzyme (CK2 alpha). The inhibitory potency of the synthesized compounds was established in a kinetic assay that used thin layer chromatography for the measurement of the rate of phosphorylation of fluorescently labelled peptide 5-TAMRA-RADDSDDDDD. The most potent inhibitor, ARC-1502 (K-i = 0.5 nM), revealed high selectivity for CK2 alpha in a panel of 140 protein kinases. Labelling of ARC-1502 with PromoFluor-647 gave the fluorescent probe ARC-1504 that possessed subnanomolar affinity towards both CK2 alpha and the holoenzyme. The probe was used in a fluorescence anisotropy-based binding assay to measure the concentration of CK2 alpha and characterize non-labelled ligands binding to the active site of CK2 alpha.