The Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China.
State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China.
J Hepatol. 2021 Mar;74(3):522-534. doi: 10.1016/j.jhep.2020.09.019. Epub 2020 Sep 25.
BACKGROUND & AIMS: 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.
目前的抗病毒疗法有助于控制乙型肝炎病毒（HBV），但无法治愈，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，迫切需要开发治愈乙型肝炎的策略。cccDNA 的功能沉默是一种重要的治疗策略，可通过靶向病毒蛋白 HBx 来实现。
我们使用 HiBiT 裂解检测系统筛选了 2000 种小分子化合物，以评估它们抑制 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，评估候选化合物的抗病毒活性及其对 cccDNA 转录的作用机制。
NAD(P)H:醌氧化还原酶 1（NQO1）抑制剂二苯并[a,j]蒽显著降低了 HBx 的表达。此外，二苯并[a,j]蒽在 HBV 感染的细胞和人源化肝脏小鼠模型中对 HBV RNA、HBV DNA、HBsAg 和 HBc 蛋白表现出强大的抗病毒活性。机制研究表明，内源性 NQO1 与 HBx 蛋白结合并保护其免受 20S 蛋白酶体介导的降解。NQO1 敲低或二苯并[a,j]蒽处理显著减少了 HBx 向 cccDNA 的募集，并抑制了 cccDNA 的转录活性，这与建立抑制性染色质状态有关。HBx 的缺失显著阻断了 NQO1 敲低或二苯并[a,j]蒽处理在 HBV 感染的细胞中诱导的抗病毒作用。
本研究报告了一种靶向 HBx 以对抗慢性 HBV 感染的新型小分子化合物；还揭示了 NQO1 通过调节 HBx 蛋白稳定性在 HBV 复制中发挥作用。
目前用于乙型肝炎的抗病毒疗法无法治愈，因为它们无法消除共价闭合环状 DNA（cccDNA），cccDNA 存在于受感染细胞的细胞核中。HBV X（HBx）蛋白在调节 cccDNA 转录中起重要作用。因此，靶向 HBx 以沉默 cccDNA 转录可能是一种重要的治疗策略。我们发现小分子二苯并[a,j]蒽可通过促进 HBx 降解来阻断 cccDNA 转录；这是治疗慢性乙型肝炎的一种很有前途的治疗策略。
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