靶向单纯疱疹病毒1型活性小分子化合物的筛选及鉴定

Screening and Identification of Active Small Molecular Compounds Targeting Herpes Simplex Virus 1

目的双向筛选抗单纯疱疹病毒1型(HSV-1)或促进HSV-1在肿瘤细胞内复制的小分子活性药物,为抗病毒药物研发或溶瘤病毒联合疗法奠定研究基础。方法通过PCR扩增、产物测序对供试病毒HSV-1 F株进行鉴定;采用TCID_(50)法测定HSV-1滴度;建立病毒核酸实时荧光定量PCR检测体系;通过细胞增殖抑制实验和实时荧光定量PCR检测体系筛选具有促进HSV-1在LN229细胞内复制活性的化合物;通过流式细胞术和病毒空斑减数实验筛选具有抗HSV-1的化合物。结果PCR扩增、产物测序结果确定供试病毒为HSV-1 F株;成功建立病毒核酸实时荧光定量PCR检测体系,灵敏度高;化合物佛司可林(FOR)可协同HSV-1抑制LN229细胞的增殖,并促进HSV-1复制相关基因ICP4、UL12和Us6的表达;化合物盐酸石蒜碱(LYC)可抑制HSV-1诱导的细胞凋亡,并减少HSV-1病毒形成空斑数、缩小空斑大小。结论FOR具有促进HSV-1复制相关基因在肿瘤细胞中表达的活性,有望成为HSV-1溶瘤联合治疗的候选药物;LYC可降低HSV-1诱导的细胞凋亡率,减少病毒空斑数,具有潜在的抗HSV-1生物学效应。

Objective To screen active small molecular drugs against herpes simplex virus type 1(HSV-1)or promoting the replication of HSV-1 in tumor cells for the development of antiviral drugs or oncolytic virus combination therapy.Methods The used HSV-1 F strain was identified by polymerase chain reaction(PCR)amplification and sequencing.The titer of HSV-1 was determined by tissue culture infectious dose 50(TCID_(50))assay.The real-time quantitative polymerasechain reaction(RT-qPCR)method was established to detect viral nucleic acid.Compounds promoting the replication activity of HSV-1 in LN229 cells were screened by cell proliferation inhibition assay and RT-qPCR method.Anti-HSV-1 compounds were screened by flow cytometry and virus plaque reducing experiment.Results PCR amplification and sequencing identified the used HSV-1 as the F strain.The RT-qPCR method for detecting HSV-1 nucleic acid was established successfully with high sensitivity.Forskolin(FOR)could cooperate with HSV-1 to inhibit the proliferation of LN229 cells and promote the expression of HSV-1 replication-related genes ICP4,UL12 and Us6.Lycorine hydrochloride(LYC)could inhibit apoptosis induced by HSV-1 and reduce the number and the size of plaques formed by HSV-1.Conclusion FOR is a potential candidate for HSV-1 oncolytic combination therapy by promoting the expression of HSV-1 replication-related genes in tumor cells.LYC can reduce apoptosis rate induced by HSV-1 and the number of viral plaques,which has potential anti-HSV-1 biological effects.