新型SOAT1蛋白靶向抑制剂的筛选与体外活性考察

Screening and in Vitro Activity of Novel Inhibitors That Targeting the SOAT1 Protein

关键词:  肝癌  SOAT1  分子对接  抑制剂  天然产物

Key words:  liver cancer  SOAT1  molecular docking  inhibitors  natural products

摘要:

目的 筛选新型甾醇O-酰基转移酶1(sterol O-acyltransferase，SOAT1)靶向抑制剂，并对其活性进行考察。方法 通过CCLE数据库筛选SOAT1高表达肝癌细胞系，选择相应细胞系作为实验细胞模型。采用Autodock Vina软件对Interbioscreen数据库中61 742个化合物与SOAT1蛋白进行分子对接，检测结合能。筛选结合力强的8个化合物，在细胞模型上用CCK8法检测其对肝癌细胞活力的影响。选择在细胞模型上活性最强的2个化合物进一步研究，并测定其IC₅₀。划痕试验及结晶紫染色法进一步检测筛选出的2个化合物对肝癌细胞迁移和增殖的影响，并采用蛋白质免疫印迹法检测化合物对肝癌细胞中SOAT1蛋白的影响。转染siRNA构建SOAT1沉默的肝癌细胞模型，并检测化合物对肝癌细胞活力的影响。结果 从CCLE数据库中筛选出SOAT1蛋白高表达的Hep3B和PLC/PRF/5肝癌细胞系作为模型细胞。通过分子对接筛选出的结合力前8的化合物中，化合物1和化合物7对上述2种肝癌细胞活力的抑制作用最为显著。此外，这2个化合物还能抑制肝癌细胞迁移及克隆形成，并降低肝癌细胞中SOAT1蛋白表达。在SOAT1沉默的肝癌细胞中，2个化合物对肝癌细胞活力的抑制作用均显著减弱。结论 化合物1和化合物7在细胞水平上能通过抑制 SOAT1 蛋白表达发挥抗肝癌作用，有潜力开发为治疗肝癌的SOAT1抑制剂。

Abstract:

OBJECTIVE To screen inhibitors that targeting sterol O-acyltransferase 1(SOAT1) protein, and to investigate the effects of the potential inhibitors in vivo. METHODS Hepatoma cells with high SOAT1 expression were screened through CCLE database and used as experimental cell models. Molecular docking between 61 742 compounds from Interbioscreen database and SOAT1 protein was performed using the Autodock Vina software, and the binding energies were calculated. Eight compounds with relative high binding energy were selected, and their effects on the viability of hepatoma cells were detected using CCK8 assays. The two most active compounds in cell models were selected for further study, and their IC₅₀ were determined. Wound healing and crystal violet staining assays were employed to detect the effects of the two compounds on the migration and proliferation of hepatoma cells. Western blotting was used to study the effects of the compounds on SOAT1 protein in hepatoma cells. siRNA were transfected into hepatoma cells to construct a SOAT1-silenced cell model, and effects of the compounds on cell viability were tested. RESULTS Hep3B and PLC/PRF/5 human hepatoma cell lines with high expression of SOAT1 protein selected from the CCLE database as model cells were screened and used as cell models. Among the 8 compounds with relative high binding affinity screened by molecular docking, Compounds 1 and 7 had the most significant inhibitory effects on the viability of the two types of liver cancer cells mentioned above. Moreover, the two compounds inhibited cell migration and cell colony formation, as well as decreased SOAT1 protein expression in hepatoma cells. In SOAT1-silenced hepatoma cells, the inhibitory effects of the two compounds on cell viability were significantly attenuated. CONCLUSION Compound 1 and compound 7 exert anti-hepatoma effects at the cellular level by inhibiting the expression of SOAT1 protein, suggesting that these two compounds have the potential to be developed into SOAT1 inhibitors for the treatment of liver cancer.