AASLD2017 [937] 杂芳基二氢嘧啶化合物GLS4 规定装配和拆卸 HBV衣

antiHBVren

Background: Heteroaryldihydropyrimidine (HAP) compounds inhibit hepatitis B virus (HBV) by interrupting the assembly of HBV core antigen (HBcAg) dimers into capsids. Effects of HAPs on the stability and function of preformed HBV capsids, however, are not wellunderstood and are being studied in this paper.
Methods:Effects of GLS4 on both assembly and disassemblyof HBV capsids were investigated by using HBV-stably-expressing HepAD38 cells and cell-free biophysical assays. Inhibitory effects of GLS4 and entecavir (ETV) on formation ofcovalently circular closed DNA (cccDNA) were evaluated in HBV-infected HepG2-NTCP cells.
Results:In HepAD38 cells, GLS4 induced perinuclear aggregation and accelerated degradation of HBcAg, followed with a highly effective blockage of capsid assembly. Notably, GLS4 treatment also caused nucleocapsid to migrate slower on native agarose gel and quick degradations of preformed capsids in cells. Further analysis of nucleocapsids (NCs) showed that GLS4 specifically caused disassembly and degradation of mature NCs containing double-stranded DNA, while immature ones containing pgRNA or single-stranded DNA were not affected. Data from cell-free biophysical assays indicated that GLS4 could directly induce the disruption of preformed capsids assembled with Cp149 proteins. HepG2-NTCP-based HBV infectious assay showed that GLS4, instead of ETV, treatment prior to virus inoculation strongly inhibited the de novo formation of cccDNA and concomitantly reduced the productionof HBsAg and HBeAg.
Conclusion: Taken together, we demonstrated in the present study that GLS4 could dually regulate both the assembly and disassembly of HBV capsids, resulting in profound inhibition of cccDNA formation that is superior to nucleot(s)ide analogues. Development of this kind of capsid inhibitors then may be an advantageous strategy to achieve better control of HBV infection.

背景：杂芳基二氢嘧啶（HAP）化合物通过中断HBV核心抗原（HBcAg）二聚体装入衣壳中来抑制乙型肝炎病毒（HBV）。然而，HAPs对预先形成的HBV衣壳的稳定性和功能的影响尚未被人们所理解，正在本文中进行研究。
方法：采用HBV稳定表达HepAD38细胞和无细胞生物物理检测方法检测GLS4对HBV衣壳的组装和拆卸的影响。在HBV感染的HepG2-NTCP细胞中评估GLS4和恩替卡韦（ETV）对形成循环闭合DNA（cccDNA）的抑制作用。
结果：在HepAD38细胞中，GLS4诱导了HBcAg的核周聚集和加速降解，其次是高效阻断衣壳组装。值得注意的是，GLS4治疗还使得核衣壳在天然琼脂糖凝胶上迁移较慢，并且在细胞中快速降解预先形成的衣壳。核衣壳（NC）的进一步分析表明，GLS4特异性引起含有双链DNA的成熟NCs的分解和降解，而含有pgRNA或单链DNA的未成熟NC不受影响。来自无细胞生物物理测定的数据表明，GLS4可以直接诱导与Cp149蛋白质组装的预先形成的衣壳的破坏。基于HepG2-NTCP的HBV感染检测显示，病毒接种前的GLS4代替ETV治疗强烈抑制了cccDNA的从头形成，并伴随地降低了HBsAg和HBeAg的产生。
结论：综合考察，本研究表明，GLS4可以双重调节HBV衣壳的组装和分解，导致cccDNA形成的深度抑制优于核苷类似物。这种衣壳抑制剂的开发可能是实现更好地控制HBV感染的有利策略。