抑制HBV核心启动子转录活性的化合物筛选与鉴定

Screening and identification of compounds inhibiting transcriptional activity of HBV core promoter

目的构建靶向核心启动子转录的稳定细胞系,筛选影响乙型肝炎病毒(hepatitis B virus,HBV)转录的药物。方法利用HepG2细胞,以重组慢病毒的方式构建由HBV核心启动子驱动的Gaussia荧光素酶稳定表达细胞系(HepG2-Pcore-Gluc)。通过检测荧光素酶活性来反映核心启动子的活性变化情况。在HBV复制细胞模型中,通过Southern blot、荧光定量PCR等进一步评估经多轮筛选得到的化合物对HBV RNA、HBV DNA的影响。结果利用HepG2-Pcore-Gluc细胞模型筛选1450种小分子化合物,第1轮筛选到41种化合物,能影响荧光素酶活性2倍以上。将这41种化合物进行第2轮剂量依赖性测试,得到2种化合物:逆转素(Reversine)、SCH58261。进一步验证发现:Reversine在瞬时转染HBV1.3倍体的HepG2和HepG2.2.15细胞中,能够有效降低HBV RNA、HBV DNA以及HBeAg、HBsAg的水平。结论建立了一种新型、高效的抗乙肝药物筛选细胞模型(HepG2-Pcore-Gluc),利用该模型筛选到一种新型分子Reversine,该分子可以通过抑制HBV核心启动子的转录活性来抑制HBV RNA的转录。

Objective To construct a stable cell line targeting the transcriptional activity of core promoter for screening drugs that affect the transcription of HBV.Methods HepG2-Pcore-Gluc was constructed by recombinant lentivirus to stably express Gaussia luciferase driven by HBV core promoter in HepG2 cells,and then the luciferase was detected to indicate the activity of the core promoter.Southern blotting,fluorescence quantitative PCR and other experiments were used to further evaluate the effects of the screened compounds on the levels of HBV RNA and HBV DNA after several rounds of screening in HBV-replication cell models.Results Forty-one compounds that could affect luciferase activity larger than 2 times were screened out of 1450 compounds in the first round screening by using HepG2-Pcore-Gluc cell model,which were tested in a second round of dose-dependent test to produce 2 compounds:Reversine and SCH58261.Further,Reversine effectively decreased the levels of HBV RNA,HBV DNA,HBeAg and HBsAg in transiently-transfected HepG2 and HepG2.2.15 cells.Conclusion Reversine is identified by using a novel cell model(HepG2-Pcore-Gluc)which targets the transcription of HBV core promoter.These results provide evidences that Reversine acts through inhibiting the transcriptional activity of core promoter.