Abstract：
Chronic hepatitis B virus (HBV) infection remains a significant global health challenge due to its link to severe conditions like HBV-related cirrhosis and hepatocellular carcinoma (HCC). Although current treatments effectively reduce viral levels, they have limited impact on certain HBV elements, namely hepatitis B surface antigen (HBsAg) and covalently closed circular DNA (cccDNA). This highlights the urgent need for innovative pharmaceutical and biological interventions that can disrupt HBsAg production originating from cccDNA. In this study, we identified a natural furanocoumarin compound, Imperatorin, which markedly inhibited the expression of HBsAg from cccDNA, by screening a library of natural compounds derived from Chinese herbal medicines using ELISA assay and qRT-PCR. The pharmacodynamics study of Imperatorin was explored on HBV infected HepG2-NTCP/PHHs and HBV-infected humanized mouse model. Proteome analysis was performed on HBV infected HepG2-NTCP cells following Imperatorin treatment. Molecular docking and bio-layer interferometry (BLI) were used for finding the target of Imperatorin. Our findings demonstrated Imperatorin remarkably reduced the level of HBsAg, HBV RNAs, HBV DNA and transcriptional activity of cccDNA both in vitro and in vivo. Additionally, Imperatorin effectively restrained the actions of HBV promoters responsible for cccDNA transcription. Mechanistic study revealed that Imperatorin directly binds to ERK and subsequently interfering with the activation of CAMP response element-binding protein (CREB), a crucial transcriptional factor for HBV and has been demonstrated to bind to the PreS2/S and X promoter regions of HBV. Importantly, the absence of ERK could nullify the antiviral impact triggered by Imperatorin. Collectively, the natural compound Imperatorin may be an effective candidate agent for inhibiting HBsAg production and cccDNA transcription by impeding the activities of HBV promoters through ERK-CREB axis.
摘要：
慢性乙型肝炎病毒（HBV）感染仍然是一个重要的全球健康挑战，因为它与HBV相关的肝硬化和肝细胞癌（HCC）等严重疾病有关。虽然目前的治疗有效降低病毒水平，他们对某些HBV元素的影响有限，即乙型肝炎表面抗原（HBsAg）和共价闭合环状DNA（cccDNA）。这凸显了对创新的药物和生物干预措施的迫切需要，这些干预措施可以破坏源自cccDNA的HBsAg生产。在这项研究中,我们发现了一种天然的呋喃香豆素化合物，Imperatorin,显着抑制HBsAg从cccDNA的表达，通过使用ELISA测定法和qRT-PCR筛选来自中草药的天然化合物库。在HBV感染的HepG2-NTCP/PHHs和HBV感染的人源化小鼠模型上探索了Imperatorin的药效学研究。在Imperatorin处理后对HBV感染的HepG2-NTCP细胞进行蛋白质组分析。分子对接和生物层干涉法（BLI）用于寻找Imperatorin的靶标。我们的研究结果表明Imperatorin显着降低HBsAg的水平，HBVRNA,HBVDNA和cccDNA的转录活性在体外和体内。此外,Imperatorin有效地抑制了负责cccDNA转录的HBV启动子的作用。机制研究表明，Imperatorin直接与ERK结合，随后干扰CAMP反应元件结合蛋白（CREB）的激活，HBV的关键转录因子，已被证明与HBV的PreS2/S和X启动子区域结合。重要的是,ERK的缺失可能使Imperatorin引发的抗病毒作用无效.总的来说，天然化合物Imperatin可能是通过ERK-CREB轴阻碍HBV启动子活性抑制HBsAg产生和cccDNA转录的有效候选药物。
