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Abstract：
Current antiviral therapies help keep HBV under control, but they are not curative, as they are unable to eliminate the intracellular viral replication intermediate termed covalently closed circular DNA (cccDNA). Therefore, there remains an urgent need to develop strategies to cure CHB. Functional silencing of cccDNA is a crucial curative strategy that may be achieved by targeting the viral protein HBx.
We screened 2,000 small-molecule compounds for their ability to inhibit HiBiT-tagged HBx (HiBiT-HBx) expression by using a HiBiT lytic detection system. The antiviral activity of a candidate compound and underlying mechanism of its effect on cccDNA transcription were evaluated in HBV-infected cells and a humanised liver mouse model.
Dicoumarol, an inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), significantly reduced HBx expression. Moreover, dicoumarol showed potent antiviral activity against HBV RNAs, HBV DNA, HBsAg and HBc protein in HBV-infected cells and a humanised liver mouse model. Mechanistic studies demonstrated that endogenous NQO1 binds to and protects HBx protein from 20S proteasome-mediated degradation. NQO1 knockdown or dicoumarol treatment significantly reduced the recruitment of HBx to cccDNA and inhibited the transcriptional activity of cccDNA, which was associated with the establishment of a repressive chromatin state. The absence of HBx markedly blocked the antiviral effect induced by NQO1 knockdown or dicoumarol treatment in HBV-infected cells.
Herein, we report on a novel small molecule that targets HBx to combat chronic HBV infection; we also reveal that NQO1 has a role in HBV replication through the regulation of HBx protein stability.
Current antiviral therapies for hepatitis B are not curative because of their inability to eliminate covalently closed circular DNA (cccDNA), which persists in the nuclei of infected cells. HBV X (HBx) protein has an important role in regulating cccDNA transcription. Thus, targeting HBx to silence cccDNA transcription could be an important curative strategy. We identified that the small molecule dicoumarol could block cccDNA transcription by promoting HBx degradation; this is a promising therapeutic strategy for the treatment of chronic hepatitis B.
We screened 2,000 small-molecule compounds for their ability to inhibit HiBiT-tagged HBx (HiBiT-HBx) expression by using a HiBiT lytic detection system. The antiviral activity of a candidate compound and underlying mechanism of its effect on cccDNA transcription were evaluated in HBV-infected cells and a humanised liver mouse model.
Dicoumarol, an inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), significantly reduced HBx expression. Moreover, dicoumarol showed potent antiviral activity against HBV RNAs, HBV DNA, HBsAg and HBc protein in HBV-infected cells and a humanised liver mouse model. Mechanistic studies demonstrated that endogenous NQO1 binds to and protects HBx protein from 20S proteasome-mediated degradation. NQO1 knockdown or dicoumarol treatment significantly reduced the recruitment of HBx to cccDNA and inhibited the transcriptional activity of cccDNA, which was associated with the establishment of a repressive chromatin state. The absence of HBx markedly blocked the antiviral effect induced by NQO1 knockdown or dicoumarol treatment in HBV-infected cells.
Herein, we report on a novel small molecule that targets HBx to combat chronic HBV infection; we also reveal that NQO1 has a role in HBV replication through the regulation of HBx protein stability.
Current antiviral therapies for hepatitis B are not curative because of their inability to eliminate covalently closed circular DNA (cccDNA), which persists in the nuclei of infected cells. HBV X (HBx) protein has an important role in regulating cccDNA transcription. Thus, targeting HBx to silence cccDNA transcription could be an important curative strategy. We identified that the small molecule dicoumarol could block cccDNA transcription by promoting HBx degradation; this is a promising therapeutic strategy for the treatment of chronic hepatitis B.
摘要：
目前的抗病毒治疗有助于保持HBV的控制，但是它们不是治愈的，因为它们无法消除称为共价闭合环状DNA（cccDNA）的细胞内病毒复制中间体。因此,仍然迫切需要制定治疗CHB的策略。cccDNA的功能沉默是可以通过靶向病毒蛋白HBx来实现的关键治疗策略。
通过使用HiBiT裂解检测系统，我们筛选了2,000种小分子化合物抑制HiBiT标记的HBx（HiBiT-HBx）表达的能力。在HBV感染的细胞和人源化肝脏小鼠模型中评估了候选化合物的抗病毒活性及其对cccDNA转录的影响的潜在机制。
Dicoumarol,NAD（P）H的抑制剂：醌氧化还原酶1（NQO1），显著降低HBx表达。此外，双香豆酚对HBVRNA显示有效的抗病毒活性，HBVDNA，HBV感染细胞中的HBsAg和HBc蛋白和人源化肝脏小鼠模型。机制研究表明，内源性NQO1结合并保护HBx蛋白从20S蛋白酶体介导的降解。NQO1敲低或双香豆素处理显着减少HBx募集到cccDNA和抑制cccDNA的转录活性，这与抑制性染色质状态的建立有关。HBx的缺乏明显阻断由NQO1敲低或双香豆素治疗在HBV感染的细胞中诱导的抗病毒作用。
这里，我们报道了一种新的小分子，靶向HBx对抗慢性HBV感染;我们还揭示了NQO1通过调节HBx蛋白稳定性在HBV复制中发挥作用。
目前的抗病毒治疗乙型肝炎是不能治愈的，因为他们无法消除共价闭合环状DNA（cccDNA），它持续存在于被感染细胞的细胞核中。HBVX(HBx)卵白在调控cccDNA转录中具有主要感化。因此，靶向HBx沉默cccDNA转录可能是一个重要的治疗策略。我们发现小分子双香豆酚可以通过促进HBx降解来阻断cccDNA转录;这是治疗慢性乙型肝炎的一种有前途的治疗策略。
通过使用HiBiT裂解检测系统，我们筛选了2,000种小分子化合物抑制HiBiT标记的HBx（HiBiT-HBx）表达的能力。在HBV感染的细胞和人源化肝脏小鼠模型中评估了候选化合物的抗病毒活性及其对cccDNA转录的影响的潜在机制。
Dicoumarol,NAD（P）H的抑制剂：醌氧化还原酶1（NQO1），显著降低HBx表达。此外，双香豆酚对HBVRNA显示有效的抗病毒活性，HBVDNA，HBV感染细胞中的HBsAg和HBc蛋白和人源化肝脏小鼠模型。机制研究表明，内源性NQO1结合并保护HBx蛋白从20S蛋白酶体介导的降解。NQO1敲低或双香豆素处理显着减少HBx募集到cccDNA和抑制cccDNA的转录活性，这与抑制性染色质状态的建立有关。HBx的缺乏明显阻断由NQO1敲低或双香豆素治疗在HBV感染的细胞中诱导的抗病毒作用。
这里，我们报道了一种新的小分子，靶向HBx对抗慢性HBV感染;我们还揭示了NQO1通过调节HBx蛋白稳定性在HBV复制中发挥作用。
目前的抗病毒治疗乙型肝炎是不能治愈的，因为他们无法消除共价闭合环状DNA（cccDNA），它持续存在于被感染细胞的细胞核中。HBVX(HBx)卵白在调控cccDNA转录中具有主要感化。因此，靶向HBx沉默cccDNA转录可能是一个重要的治疗策略。我们发现小分子双香豆酚可以通过促进HBx降解来阻断cccDNA转录;这是治疗慢性乙型肝炎的一种有前途的治疗策略。
