目的：评价以酵母细胞为模式菌高通量筛选Pin1抑制剂的方法。方法以“生型酵母菌W303-1a和温度敏感型突变株L94P为模式菌，筛选Pin1抑制剂；四甲基偶氮唑盐(MTT)比色法检验候选化合物对肿瘤细胞的杀伤作用；体外酶学实验验证候选化合物对Pin1的抑制作用；流式细胞仪检测候选化合物对肿瘤细胞周期和凋亡的阻滞和诱导作用；免疫印迹(Western Blotting)分析候选化合物对肿瘤细胞中Cyclin D1和Pin1表达量的影响。结果候选化合物显示出对温度敏感型突变株的特异性抑制作用；MTT实验证实化合物能抑制肿瘤细胞的增殖；酶学实验和Western Blotting共同阐释了化合物对重组纯化的人Pin1具抑制作用但并不影响Pin1蛋白的表达；流式结果表明化合物能诱导细胞发生凋亡和G0/G1期阻滞；化合物还能抑制肿瘤细胞中Cyclin D1的蛋白表达并呈剂量依赖性。结论首次发现Pin1为苯并呋喃衍生物抗肿瘤作用的靶点之一。%Objective To evaluate potential small molecular inhibitors through high throughput screening (HTS) using budding yeast cells. Methods In vitro biochemical assay using purified recombinant human Pin1 was performed to test the inhibition of candidate determined by HTS using wild-type yeast cells and L94P temperature sensitive mutant. MTT proliferation assay was carried out to detect ZY0625’s cellular toxicity. Flow cytometric analysis was operated to assess ZY0625’s influence on cell cycle progression and apoptosis. Western Blotting was performed to determine the effects of candidate on Pin1 and Cyclin D1 expression. Results ZY0625 inhibited the growth of L94P temperature sensitive mu-tant more strongly than wild-type yeast cells, moreover ZY0625 inhibited purified recombinant human Pin1 in v itro without influencing the expression of Pin1 in tumor cells. Cell culture based tests of ZY0625 activity showed that ZY0625 potently inhibited the proliferation of human cancer cell lines. Flow cytometry confirmed that ZY0625 could induce apoptosis of tumer cells and modulate cell cycle distribution moderately with an elevated percentage of cells in G0/G1, as well. Like other Pin1 inhibitors, ZY0625 increased the protein expression of Cyclin D1 in a dose dependent fashion. Conclusion Pin1 is for the first time verified as one of the targets that contributes to the benzofuran deriva-tives' anticancer activity.
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