定靶突变温敏酵母模型在Pin1抑制剂筛选中的应用

Application of temperature sensitive yeast models with definite target in the screening of potential human Pin1 inhibitors

利用定靶突变温敏酵母模型寻找Pin1的新型抑制剂,并探讨阳性化合物的体内外抗肿瘤活性。采用MTT法、流式细胞仪法、免疫印迹法、划痕实验和虚拟对接检测阳性化合物的体外活性。利用移植瘤裸鼠模型检测阳性化合物的体内活性。结果发现,酵母模型初筛得到的阳性化合物8-11在体外,对Pin1的IC50值为(10.40±1.68)μmol·L-1,能抑制多种肿瘤细胞的增殖,诱导HeLa细胞G1期阻滞和凋亡,降低Cyclin D1的蛋白水平表达,降低肿瘤细胞迁移能力,虚拟对接中与Pin1的活性结构域呈现出较高的亲和性;在体内,8-11抑制裸鼠移植瘤生长。本研究首次证实8-11可通过抑制Pin1发挥抗肿瘤作用。

This study is to explore new lead compounds by inhibition of Pinl for anticancer therapy using temperature sensitive mutants. As Pinl is conserved from yeast to human, we established a high-throughput screening method for Pinl inhibitors, which employed yeast assay. This method led to the identification of one potent hits, 8-11. In vitro, 8-11 inhibited purified Pin1 enzyme activity with IC50 of （10.40±1.68）μmol·L-1, induced G1 phase arrest and apoptosis, showed inhibitory effects on a series of cancer cell proliferation, reduced Cyclin D1 expression, was defined as reciprocally matched for protein-ligand complex in virtual docking analysis and reduced cell migration ability. In vivo, we could observe reduction of tumor volume after treatment with 8-11 in xenograft mice compared with vehicle DMSO treatment. Altogether, these results provide for the first time the involvement of 8-11 in the anticancer activity against Pinl.