Table3_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（Casein Kinase 1）家族通过调控多条信号通路参与多种生理过程，包括Wnt/β-连环蛋白、刺猬（Hedgehog）以及p53信号通路。现有研究证实，各类激酶尤其是CK1的突变或表达失调，会促进癌症、神经退行性疾病与炎症的发生发展。越来越多的证据显示，具备CK1同工型特异性的小分子抑制剂——包括与苯并咪唑类化合物结构相似、基于Wnt生成抑制剂（IWP）的CK1δ和CK1ε特异性抑制剂——展现出极具潜力的治疗应用价值。

方法：本研究评估了斑马鱼模型系统用于这类CK1抑制剂评价的适用性。为此，我们比较了人类CK1同工型与斑马鱼同源蛋白的动力学参数。此外，还在体内分析了选择性CK1δ抑制对斑马鱼胚胎发育的影响。

结果：研究结果表明，化合物G2-2对斑马鱼CK1δA与CK1δB的抑制效果可媲美人类CK1δ：其对斑马鱼CK1δA、CK1δB的半最大抑制浓度（IC50）分别为345 nM和270 nM，人类CK1δ的IC50则为503 nM；而化合物G2-3对斑马鱼CK1δA、B的IC50分别为514 nM和561 nM，人类CK1δ的IC50为562 nM。进一步的体内实验显示，选择性CK1δ抑制会诱导斑马鱼胚胎出现表型异常，提示CK1δ被下调。使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抑制生物学相关性初步研究的体内实验模型系统。