酵母模型在Aurora-B激酶抑制剂筛选中的应用

Screening of potential small-molecular inhibitor of human Aurora-B kinase using budding yeast

目的以酵母细胞为模式菌高通量筛选微生物来源的Aurora-B激酶抑制剂。方法以野生型酵母菌Y300和ipl1-321温度敏感型突变株为模式菌,筛选微生物来源的Aurora-B激酶抑制剂;体外酶学实验验证候选化合物对Aurora-B激酶的抑制作用;Western Blotting分析候选化合物对肿瘤细胞中histone H3磷酸化的影响。结果Jadomycin B显示出对ipl1-321温度敏感型突变株的特异性抑制作用;体外酶学实验证实jadomycin B对重组纯化的人Aurora-B激酶具抑制作用;jadomycin B能抑制多种肿瘤细胞中histone H3的磷酸化并呈剂量依赖性。结论以Y300和ipl1-321酵母细胞为高通量筛选模型筛选到一个新的Aurora-B激酶抑制剂jadomycin B。

Objective To find potential small-molecular inhibitors from microbial natural products through high throughput screening （HTS） using budding yeast cells. Methods In vitro biochemical assay was performed to test the inhibition of candidates determined by HTS using wild-type yeast ceils and ipll-321 temperature sensitive mutant on purified recombinant human Aurora-B kinase. Western Blotting was performed to determine the effect of candidates on the phosphorylation of histone H3 on Ser10. Result Jadomycin B inhibited the growth of ipl1-321 temperature sensitive mutant more strongly than wild-type yeast cells, moreover jadomycin B inhibited both purified recombinant human Aurora-B kinase in vitro and phosphorylation of histone H3 on Ser10 in tumor cells in a dose-dependent manner. Conclusion Jadomycin B was determined as a new inhibitor of Aurora-B through HTS using wild-type yeast cells and ipl1-321 temperature sensitive mutant.