Table6_Zebrafish as model system for the biological characterization of CK1 inhibitors.DOCX

Introduction: The CK1 family is involved in a variety of physiological processes by regulating different signaling pathways, including the Wnt/β-catenin, the Hedgehog and the p53 signaling pathways. Mutations or dysregulation of kinases in general and of CK1 in particular are known to promote the development of cancer, neurodegenerative diseases and inflammation. There is increasing evidence that CK1 isoform specific small molecule inhibitors, including CK1δ- and CK1ε-specific inhibitors of Wnt production (IWP)-based small molecules with structural similarity to benzimidazole compounds, have promising therapeutic potential.
Methods: In this study, we investigated the suitability of the zebrafish model system for the evaluation of such CK1 inhibitors. To this end, the kinetic parameters of human CK1 isoforms were compared with those of zebrafish orthologues. Furthermore, the effects of selective CK1δ inhibition during zebrafish embryonic development were analyzed in vivo.
Results: The results revealed that zebrafish CK1δA and CK1δB were inhibited as effectively as human CK1δ by compounds G2-2 with IC50 values of 345 and 270 nM for CK1δA and CK1δB versus 503 nM for human CK1δ and G2-3 exhibiting IC50 values of 514 and 561 nM for zebrafish CK1δA and B, and 562 nM for human CK1δ. Furthermore, the effects of selective CK1δ inhibition on zebrafish embryonic development in vivo revealed phenotypic abnormalities indicative of downregulation of CK1δ. Treatment of zebrafish embryos with selected inhibitors resulted in marked phenotypic changes including blood stasis, heart failure, and tail malformations.
Conclusion: The results suggest that the zebrafish is a suitable in vivo assay model system for initial studies of the biological relevance of CK1δ inhibition.

引言：CK1（酪蛋白激酶1）家族通过调控多条信号通路参与多种生理过程，涵盖Wnt/β-连环蛋白、Hedgehog以及p53信号通路。目前已知，激酶（尤其是CK1）的突变或表达失调会促进癌症、神经退行性疾病以及炎症的发生发展。越来越多的证据表明，靶向CK1亚型的小分子抑制剂——包括结构与苯并咪唑类化合物相似的、基于Wnt生成抑制剂（IWP）的CK1δ与CK1ε特异性抑制剂——具有可观的治疗潜力。
方法：本研究旨在评估斑马鱼模型体系用于此类CK1抑制剂评价的适用性。为此，我们将人源CK1亚型的动力学参数与其斑马鱼同源物进行了对比。此外，我们还在体内分析了选择性抑制CK1δ对斑马鱼胚胎发育的影响。
结果：研究结果显示，化合物G2-2对斑马鱼CK1δA与CK1δB的抑制效果与对人源CK1δ的抑制效果相当：CK1δA和CK1δB的半抑制浓度（IC50）分别为345 nM与270 nM，而人源CK1δ的IC50为503 nM；化合物G2-3对斑马鱼CK1δA、CK1δB的半抑制浓度分别为514 nM与561 nM，对人源CK1δ的半抑制浓度为562 nM。此外，体内选择性抑制CK1δ对斑马鱼胚胎发育的影响实验显示，出现了提示CK1δ表达下调的表型异常。用筛选得到的抑制剂处理斑马鱼胚胎后，可观察到显著的表型改变，包括血液淤滞、心力衰竭以及尾部畸形。
结论：本研究结果表明，斑马鱼可作为适用于CK1δ抑制作用生物学相关性初步研究的体内实验模型体系。