Inhibition of Hepatitis B Virus Replication by Rheum palmatum L. Ethanol Extract in a Stable HBV-producing Cell

Hepatitis B virus(HBV) infection is a severe health problem in the world.However,there is still not a satisfactory therapeutic strategy for the HBV infection.To search for new anti-HBV agents with higher efficacy and less side-effects,the inhibitory activities of traditional Chinese medicine Rheum palmatum L.ethanol extract(RPE) against HBV replication were investigated in this study.Quantitative real-time polymerase chain reaction(PCR) was employed to analyze the inhibitory activity of RPE against HBV-DNA replication in a stable HBV-producing cell line HepAD38; the expression levels of HBV surface antigen(HBsAg) and e antigen(HBeAg) were also determined by enzyme linked immunosorbent assay(ELISA) after RPE treatment.RPE could dose-dependently inhibit the production of HBV-DNA and HBsAg.The concentration of 50% inhibition(IC50) was calculated at 209.63,252.53 μg/mL,respectively.However,its inhibitory activity against HBeAg expression was slight even at high concentrations.RPE had a weak cytotoxic effect on HepAD38 cells(CC50 = 1 640 μg/mL) and the selectivity index(SI) was calculated at 7.82.Compared with two anthraquinone derivatives emodin and rhein,RPE showed higher ability of anti-HBV and weaker cytotoxicity.So Rheum palmatum L.might possess other functional agents which could effectively inhibit HBV-DNA replication and HBsAg expression.Further purification of the active agents,identification and modification of their structures to improve the efficacy and decrease the cytotoxicity are required.     

蒽醌对HBV稳定产毒细胞株HepAD38HBV DNA复制的抑制作用

目的对掌叶大黄乙醇提取物中天然成份-蒽醌类化合物的抑制乙肝病毒（HBV）的活性进行检测。方法制备掌叶大黄乙醇提取物（RPE）并从中分离提取有效成份,荧光定量PCR法和ELISA法检测PRE及其各成份对HepAD38细胞中HBV表达及复制的作用;同时用MTY法检测细胞毒性。结果从掌叶大黄乙醇提取物中分离得到的大黄酚-8-O-β-D-葡萄糖甙对HBVDNA复制以及抗原表达均有明显的抑制活性。同时MTY法检测显示大黄酚-8-O-β-D-葡萄糖甙没有明显毒副作用。结论大黄酚-8-O-β-D-葡萄糖甙可以有效抑制HBVDNA复制及HBsAg表达,对其进一步的研究将有助于在此化合物基础上开发出新的抗HBV药物。     

Inhibition of the replication of hepatitis B virus in vitro by a novel 2,6-diaminopurine analog, beta-LPA

Antiviral therapy of chronic hepatitis B remains a major clinical problem worldwide. Like lamivudine, nucleoside analogs have become the focus of investigation of anti-hepatitis B virus (anti-HBV) drugs. Here, β-LPA is a novel 2,6-diaminopurine analog found to possess potent anti-HBV activity. In HepG2.2.15 cell line, β-LPA had a 50% effective concentration (EC₅₀) of 0.01 μM against HBV, as determined by analysis of secreted and intracellular episomal HBV DNA. Levels of HBV surface antigen (HBsAg) and e antigen (HBeAg) in drug-treated cultures revealed that β-LPA had no significant inhibitory effects on HBsAg and HBeAg. β-LPA didn’t show any cytotoxicity up to 0.4 μM with a 50% cytotoxic concentration (CC₅₀) of 50 μM. Furthermore, treatment with β-LPA resulted in no apparent inhibitory effects on mitochondrial DNA content. Considering the potent inhibition of HBV DNA synthesis and no obvious toxicity of β-LPA, this compound should be further explored for development as an anti-HBV drug.     

Synthesis,structure–activity relationships and biological evaluation of dehydroandrographolide and andrographolide derivatives as novel anti-hepatitis B virus agents

Dehydroandrographolide and andrographolide, two natural diterpenoids isolated from Andrographis paniculata possessed activity against HBV DNA replication with IC₅₀ values of 22.58 and 54.07 μM and low SI values of 8.7 and 3.7 in our random assay. Consequently, 48 derivatives of dehydroandrographolide and andrographolide were synthesized and evaluated for their anti-HBV properties to yield a series of active derivatives with lower cytotoxicity, including 14 derivatives against HBsAg secretion, 19 derivatives against HBeAg secretion and 38 derivatives against HBV DNA replication. Interestingly, compound 4e could inhibit not only HBsAg and HBeAg secretions but also HBV DNA replication with SI values of 20.3, 125.0 and 104.9. Furthermore, the most active compound 2c with SI value higher than 165.1 inhibiting HBV DNA replication was revealed with the optimal log P value of 1.78 and log D values. Structure–activity relationships (SARs) of the derivatives were disclosed for guiding the future research toward the discovery of new anti-HBV drugs.     

Synthesis, structure-activity relationships and biological evaluation of caudatin derivatives as novel anti-hepatitis B virus agents

A series of caudatin derivatives were synthesized, and their anti-hepatitis B virus (HBV) activity was evaluated in HepG 2.2.15 cells. Most of the 3-O-substituted caudatin derivatives showed effective anti-HBV activity. Among the tested compounds, six compounds (2e-2h, 2l, 2r) exhibited significantly inhibitory activity against HBV DNA replication with IC(50) values in the range of 2.82-7.48 μM. Interestingly, two compounds (2e, 2f) had potent activity inhibiting not only the secretion of HBsAg (IC(50)=18.68 μM, 21.71 μM), HBeAg (IC(50)=13.16 μM, 33.73 μM), but also HBV DNA replication (IC(50)=7.48 μM, 3.63 μM). The structure-activity relationships (SARs) of caudatin derivatives had been discussed, which were useful for caudatin derivatives to be explored and developed as novel anti-HBV agents.     

Design and Synthesis of (3-Phenylisoxazol-5-yl)methanimine Derivatives as Hepatitis B Virus Inhibitors

Series of (3-phenylisoxazol-5-yl)methanimine derivatives were synthesized, and evaluated for anti-hepatitis B virus (HBV) activity in vitro. Half of them more effectively inhibited HBsAg than 3TC, and more favor to inhibit secretion of HBeAg than to HBsAg. Part of the compounds with significant inhibition on HBeAg were also effectively inhibit replication of HBV DNA. Compound (E)-3-(4-fluorophenyl)-5-((2-phenylhydrazineylidene)methyl)isoxazole inhibited excellently HBeAg with IC₅₀ in 0.65 μM (3TC(Lamivudine) in 189.90 μM), inhibited HBV DNA in 20.52 μM (3TC in 26.23 μM). Structures of compounds were determined by NMR and HRMS methods, and chlorination on phenyl ring of phenylisoxazol-5-yl was confirmed by X-ray diffraction analysis, and the structure–activity relationships (SARs) of the derivatives was discussed. This work provided a new class of potent non-nucleoside anti-HBV agents.     

Effects of 1,2,4,6-<Emphasis Type="Italic">tetra</Emphasis>-O-galloyl-β-D-glucose from <Emphasis Type="Italic">P. emblica</Emphasis> on HBsAg and HBeAg secretion in HepG2.2.15 cell culture

A polyphenolic compound, 1,2,4,6-tetra-O-galloyl-β-D-glucose (1246TGG), was isolated from the traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae) and assayed for its potential as an anti-hepatitis B virus (HBV) agent. The cytotoxicity of 1246TGG on HepG2.2.15 as well as HepG2 cells was determined by observing cytopathic effects, and the effects of 1246TGG on secretion of HBsAg and HBeAg in HepG2.2.15 cells were assayed by enzyme immunoassay. Results indicates that treatment with 1246TGG (6.25 μg/mL, 3.13 μg/mL), reduced both HBsAg and HBeAg levels in culture supernatant, yet the inhibitory effects tend to decline with the assay time. This study provides a basis for further investigation of the anti-HBV activity and possible mechanism of action of 1246TGG.     

siRNA Combinations Mediate Greater Suppression of Hepatitis B virus Replication in Mice

Hepatitis B virus (HBV) infection is a major world-wide health problem. The major obstacles for current anti-HBV therapy are the low efficacy and the occurrence of drug resistant HBV mutations. Recent studies have demonstrated that combination therapy can enhance antiviral efficacy and overcome shortcomings of established drugs. In this study, the inhibitory effect mediated by combination of siRNAs targeting different sites of HBV in transgenic mice was analyzed. HBsAg and HBeAg in the sera of the mice were analyzed by enzyme-linked immunoadsorbent assay, HBV DNA by real-time PCR and HBV mRNA by RT-PCR. Our data demonstrated that all the three siRNAs employed showed marked anti-HBV effects. The expression of HBsAg and the replication of HBV DNA could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, combination of siRNAs compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication, even though the final concentration of siRNA used for therapy was the same. Secreted HBsAg and HBeAg in the serum of mice treated with siRNA combination were reduced by 96.7 and 96.6 %, respectively. Immunohistochemical detection of liver tissue revealed 91 % reduction of HBsAg-positive cells in the combination therapy group. The combination of siRNAs caused a greater inhibition in the levels of viral mRNA and DNA (90 and 87.7 %) relative to the control group. It was noted that the siRNA3 showed stronger inhibition of cccDNA (78.6 %). Our results revealed that combination of siRNAs mediated a stronger inhibition of viral replication and antigen expression in transgenic mice than single siRNAs.     

从干扰素诱导蛋白MxA筛选抗乙肝病毒活性肽

黏病毒抗性蛋白A（myxovirus resistance protein A,MxA）是由干扰素诱导的具有重要抗乙肝病毒（hepatitis B virus,HBV）功能的蛋白质,我们前期工作发现,MxA主要依赖其中心互作结构域（central interactive domain,CID）与病毒直接相互作用发挥功能,但其具体的抗病毒功能区以及功能区是否具有独立的抗病毒活性仍不清楚。本研究拟鉴定MxA蛋白上的抗乙肝病毒活性肽。首先从全长MxA构建缺失突变体ΔCID和截短体CID,以HepG2-2-15细胞为病毒模型,分别转染空载质粒、MxA、ΔCID和CID,免疫荧光法检测转染效率,Western印迹法检测质粒表达,酶联免疫法测定细胞培养液中HBsAg、HBeAg的量及荧光定量PCR法测定乙肝病毒 DNA的量,评估CID段的抗乙肝病毒活性。根据CID段的晶体结构,缩短肽段长度,构建α1、α2、α3等9段肽段质粒,鉴定各段的抗乙肝病毒活性和细胞毒性（MTT法）。运用计算生物学手段--分子对接法预测MxA蛋白与病毒相互作用的模式和位点。结果显示,ΔCID、CID和9段肽段质粒的序列及表达正确,9段肽段的表达量未见显著性差异,无显著的细胞毒性。CID组和黏病毒抗性蛋白A组较对照组乙肝病毒的复制水平显著降低,CID组细胞上清中HBsAg、HBeAg及乙肝病毒 DNA的量分别减少了55.57%±8.48%、68.37%±6.24%、66.67%±6.40%,P＜0.01;MxA组的3个指标分别减少了61.63%±3.11%、70.77%±7.25%、73.73%±6.18%,P＜0.01;ΔCID组较对照组无明显变化。9段肽段中α1组较对照组HBsAg、HBeAg及乙肝病毒 DNA的量有显著下降,分别减少了48.33%±1.70%、70.67%±3.30%、68.95%±2.55%,P＜0.001,表明α1对乙肝病毒具有强抑制活性。分子对接的结果显示,384 ～ 408位氨基酸是MxA蛋白与病毒互作的关键位点,该区域落在α1肽段上,验证了α1是MxA蛋白抗乙肝病毒及与乙肝病毒相互作用中的关键区段。本研究筛选并鉴定出人干扰素诱导蛋白MxA上最有效的抗乙肝病毒活性肽α1,研究结果为抗乙肝病毒多肽类新药的研发奠定了基础。     

Formation of covalently closed circular DNA in Hep38.7-Tet cells,a tetracycline inducible hepatitis B virus expression cell line

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in chronic HBV infection. However, analysis of the molecular mechanism of cccDNA formation is difficult because of the low efficiency in tissue cultured cells. In this study, we developed a more efficient cccDNA expression cell, Hep38.7-Tet, by subcloning from a tetracycline inducible HBV expression cell, HepAD38. Higher levels of cccDNA were produced in Hep38.7-Tet cells compared to HepAD38 cells. In Hep38.7-Tet cells, the cccDNA was detectable at six days after HBV induction. HBV e antigen (HBeAg) secretion was dependent upon cccDNA production. We screened chemical compounds using Hep38.7-Tet cells and HBeAg secretion as a marker. Most of the hit compounds have already been reported as anti-HBV compounds. These data suggested that Hep38.7-Tet cells will be powerful tools for analysis of the molecular mechanism of cccDNA formation/maintenance and development of novel therapeutic agents to control HBV infection.     

Mucroporin-M1 Inhibits Hepatitis B Virus Replication by Activating the Mitogen-activated Protein Kinase (MAPK) Pathway and Down-regulating HNF4α in Vitro and in Vivo

Hepatitis B virus (HBV) is a noncytopathic human hepadnavirus that causes acute, chronic hepatitis and hepatocellular carcinoma (HCC). As the clinical utility of current therapies is limited, new anti-HBV agents and sources for such agents are still highly sought after. Here, we report that Mucroporin-M1, a scorpion venom-derived peptide, reduces the amount of extracellular HBsAg, HBeAg, and HBV DNA productions of HepG2.2.15 cells in a dose-dependent manner and inhibits HBV capsid DNA, HBV intracellular RNA replication intermediates and the HBV Core protein in the cytoplasm of HepG2.2.15 cells. Using a mouse model of HBV infection, we found that HBV replication was significantly inhibited by intravenous injection of the Mucroporin-M1 peptide. This inhibitory activity was due to a reduction in HBV promoter activity caused by a decrease in the binding of HNF4α to the precore/core promoter region. Furthermore, we confirmed that Mucroporin-M1 could selectively activate mitogen-activated protein kinases (MAPKs) and lead to the down-regulation of HNF4α expression, which explains the decreased binding of HNF4α to the HBV promoter. Moreover, when the protein phosphorylation activity of the MAPK pathway was inhibited, both HNF4α expression and HBV replication recovered. Finally, we proved that treatment with the Mucroporin-M1 peptide increased phosphorylation of the MAPK proteins in HBV-harboring mice. These results implicate Mucroporin-M1 peptide can activate the MAPK pathway and then reduce the expression of HNF4α, resulting in the inhibition of HBV replication in vitro and in vivo. Our work also opens new doors to discovering novel anti-HBV agents or sources.     

Synthesis and biological assay of 4-aryl-6-chloro-quinoline derivatives as novel non-nucleoside anti-HBV agents

A series of 4-aryl-6-chloro-quinoline derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities, namely the abilities to inhibit the secretion of HBV surface antigen (HBsAg), HBV e antigen (HBeAg), and replication of HBV DNA in HepG 2.2.15 cells. Most of the compounds exhibited moderate inhibitory activity against the secretion of HBsAg and HBeAg. Nine compounds (3, 5, 6, 7, 10, 14, 17, 20, 24) showed significant inhibition against HBV DNA replication with IC(50) values in the range of 4.4-9.8 μM, which were comparative to that of positive control tenofovir. Of them, compounds 10, 17, and 20 had low cytotoxicities, resulting in high SI values, >551.2, >143.7, and >284.5, respectively.     

乙肝病毒载量与血清标志物及ALT相关性研究

探讨了乙型肝炎病毒核酸(HBV-DNA)水平与乙型肝炎免疫标志物(HBVM)和丙氨酸氨基转移酶(ALT)的关系。分别采用实时荧光定量聚合酶链反应(FQ-PCR),酶联免疫法和连续监测法检测了345例血清标本HBV-DNA含量,HBVM(HBsAg、HBsAb、HBeAg、HBeAb、抗HBcIgM)表达及ALT水平。HBsAg、HBeAg(和抗HBcIgM)阳性患者HBV DNA阳性率要明显高于HBsAg、HBeAb(和抗HBcIgM)阳性患者、仅HBsAg阳性患者及HBsAb、HBeAb阳性患者(P<0.01)。血清HBeAg阳性标本HBV-DNA阳性率为98.7%,明显高于HBeAg阴性标本的61.6%(P<0.01),并且血清HBeAg阳性标本HBV-DNA含量(log值,7.42±1.43)也明显高于HBeAg阴性标本(4.36±1.73)(P<0.01);在HBV-DNA含量小于107copy/mL的标本中,ALT与HBV-DNA含量呈正相关(P<0.01)。血清中HBV DNA含量与乙型肝炎免疫标志物以及肝细胞损伤三者之间存在密切的关系,在临床工作中应对血清HBVM、ALT和HBV-DNA含量联合检测,这样才能更准确地判断患者病情、预后及指导抗病毒药物的应用。     

Anti-HBV agents. Part 1: Synthesis of alisol A derivatives: a new class of hepatitis B virus inhibitors

A series of alisol A derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities and cytotoxicities in vitro. The preliminary investigation demonstrates that simple modifications of the parent structure of alisol A can produce a number of potentially important derivatives against HBV. The most active anti-HBV compound 6a showed high activities against the secretion of HBV surface antigen (IC(50)=0.024 mM), HBV e antigen (IC(50)=0.028 mM) and remarkable selective indices (SI(HBsAg)>108, SI(HBeAg)>93), which was selected for further evaluation as a novel HBV inhibitor.     

Design,synthesis and biological evaluation of seco-A-pentacyclic triterpenoids-3,4-lactone as potent non-nucleoside HBV inhibitors

A series of seco-A-pentacyclic triterpenoids-3,4-lactone were synthesized and the anti-HBV activities were evaluated in vitro. Several compounds inhibited the secretion of HBV antigen and the replication of HBV DNA in micromolar level. Compounds D7 and D10, seco-A-oleanane-3,4-lactone, suppressed the HBeAg secretion with IC₅₀ values of 0.14?μM and 0.86?μM respectively, and the inhibitory activities were also confirmed by detecting the fluorescence intensity of FITC-labeled monoclonal mouse HBeAg antibody via flow cytometry. Compounds D7 and D10 as well as B4, ring-A cleaved 3,30-dioic acid, also displayed remarkable inhibition on both HBV DNA replication at the concentration of 25?μM and HBV cccDNA (covalently closed circularDNA) replication with IC₅₀ values of 33.5?μM, 32.7?μM and 12.3?μM respectively.     

Selective inhibition of hepatitis B virus replication by RNA interference

Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells including genes of viral origin. To evaluate the therapeutic efficacy of siRNA against the hepatitis B virus (HBV), we studied the effect of transfection of the HBV-inducible cell lines HepAD38 and HepAD79 with siRNA specific for the core gene of the HBV genome. HepAD38 cells produce wild-type HBV, whereas HepAD79 cells produce the lamivudine resistant YMDD variant. Transfection of HepAD38 cells with either 1.6 or 4 microg/ml siRNA resulted in a profound inhibition (72% and 98%, respectively) of viral replication (as assessed by real-time quantitative PCR). The inhibitory effect was corroborated by a marked reduction of HBV core protein synthesis in induced HepAD38 cells. In HepAD79 cells, transfected with 1.6 or 4 microg/ml HBV-specific siRNA, virus production was reduced by 75% and 89%, respectively.     

Schisanwilsonins A–G and related anti-HBV lignans from the fruits of Schisandra wilsoniana

Seven new dibenzocyclooctane lignans, schisanwilsonins A–G (1–7), were isolated from the fruits of Schisandra wilsoniana, together with five known lignans (8–12). The structures of these new compounds were elucidated by spectroscopic methods including 2D-NMR techniques. The 12 lignans were tested for anti-hepatitis B virus (HBV) activity in vitro. Schisanwilsonin D (4), schisantherin C (9), deoxyschizandrin (10) and (+)-gomisin K₃ (11) showed anti-HBV activity. 9 exhibited the most potent anti-HBV activity with potency against HBsAg and HBeAg secretion by 59.7% and 34.7%, respectively, at 50 μg/mL.