Identification of Disubstituted Sulfonamide Compounds as Specific Inhibitors of Hepatitis B Virus Covalently Closed Circular DNA Formation
ABSTRACT Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in viral infection and persistence and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment. Therefore, there is an urgent need for the development of novel therapeutic agents that directly target cccDNA formation and maintenance. By employing an innovative cell-based cccDNA assay in which secreted HBV e antigen is a cccDNA-dependent surrogate, we screened an in-house small-molecule library consisting of 85,000 drug-like compounds. Two structurally related disubstituted sulfonamides (DSS), termed CCC-0975 and CCC-0346, emerged and were confirmed as inhibitors of cccDNA production, with low micromolar 50% effective concentrations (EC(50)s) in cell culture. Further mechanistic studies demonstrated that DSS compound treatment neither directly inhibited HBV DNA replication in cell culture nor reduced viral polymerase activity in the in vitro endogenous polymerase assay but synchronously reduced the levels of HBV cccDNA and its putative precursor, deproteinized relaxed circular DNA (DP-rcDNA). However, DSS compounds did not promote the intracellular decay of HBV DP-rcDNA and cccDNA, suggesting that the compounds interfere primarily with rcDNA conversion into cccDNA. In addition, we demonstrated that CCC-0975 was able to reduce cccDNA biosynthesis in duck HBV-infected primary duck hepatocytes. This is the first attempt, to our knowledge, to identify small molecules that target cccDNA formation, and DSS compounds thus potentially serve as proof-of-concept drug candidates for development into therapeutics to eliminate cccDNA from chronic HBV infection.
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ABSTRACT: The persistence of hepatitis B virus (HBV) infection is maintained by the nuclear viral covalently closed circular DNA (cccDNA), which serves as transcription template for viral mRNAs. Previous studies suggested that cccDNA contains methylation-prone CpG islands, and that the minichromosome structure of cccDNA is epigenetically regulated by DNA methylation. However, the regulatory effect of each CpG island methylation on cccDNA activity remains elusive. In the present study, we analyzed the distribution of CpG methylation within cccDNA in patient samples and investigated the impact of CpG island methylation on cccDNA-driven virus replication. Our study revealed the following observations: 1) Bisulfite sequencing of cccDNA from chronic hepatitis B patients indicated that CpG island I was seldom methylated, 2) CpG island II methylation was correlated to the low level of serum HBV DNA in patients, and in vitro methylation studies confirmed that CpG island II methylation markedly reduced cccDNA transcription and subsequent viral core DNA replication, 3) CpG island III methylation was associated with low serum HBsAg titers, and 4) Furthermore, we found that HBV genotype, HBeAg positivity, and patient age and liver fibrosis stage were also relevant to cccDNA CpG methylation status. Therefore, we clearly demonstrated that the status of cccDNA methylation is connected to the biological behavior of HBV. Taken together, our study provides a complete profile of CpG island methylation within HBV cccDNA and new insights for the function of CpG methylation in regulating HBV cccDNA transcription.PLoS ONE 10/2014; 9(10):e110442. DOI:10.1371/journal.pone.0110442 · 3.53 Impact Factor
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ABSTRACT: To investigate the reduction in hepatitis B virus (HBV) covalently closed-circular DNA (cccDNA) with entecavir (ETV) or lamivudine (LAM). This analysis included patients who had participated in the randomized Phase III study ETV-022 comparing ETV vs LAM in nucleos(t)ide-naive, HBeAg-positive patients. Patients received ETV (0.5 mg daily) or LAM (100 mg daily) for a minimum of 52 wk. Patients were eligible to participate in this sub-study if they had paired biopsies at baseline and week 48 with evaluable measurements for hepatic HBV cccDNA and total hepatic HBV DNA. The main objective was to compare changes in hepatic HBV cccDNA and total hepatic HBV DNA at week 48 of ETV or LAM treatment, which was a secondary endpoint of study ETV-022. Additional post hoc analyses included linear regression analyses to assess associations of baseline levels and on-treatment changes of cccDNA with other baseline factors [sex, age, serum HBV DNA, alanine aminotransferase (ALT), Knodell necroinflammatory score, Ishak fibrosis score, total hepatic HBV DNA, and HBV genotype], or on-treatment factors (changes from baseline at week 48 in serum HBV DNA, ALT, Knodell necroinflammatory score, Ishak fibrosis score, total hepatic HBV DNA, and HBeAg loss at week 48). Overall, 305 patients (ETV = 159; LAM = 146) of ETV-022 had paired baseline and week 48 liver biopsies with evaluable measurements for hepatic HBV cccDNA and total hepatic HBV DNA, and were included in this analysis. Baseline demographics and disease characteristics were comparable between the two arms. After 48 wk, ETV resulted in significantly greater reductions in hepatic HBV cccDNA [-0.9 log10 copies/human genome equivalent (HGEq) vs -0.7 log10 copies/HGEq; P = 0.0033] and total hepatic DNA levels (-2.1 log10 copies/HGEq vs -1.6 log10 copies/HGEq; P < 0.0001) than LAM. Virologic, biochemical, and histologic response rates at week 48 were also greater with ETV than with LAM. Baseline HBV cccDNA levels were positively associated with baseline levels of serum HBV DNA and total hepatic HBV DNA, and negatively associated with HBV genotype F. On-treatment changes in HBV cccDNA levels were negatively associated with baseline levels of serum HBV DNA and baseline ALT, and were positively associated with on-treatment changes in the levels of serum HBV DNA, total hepatic HBV DNA levels, and ALT, change in Knodell necroinflammatory score, and HBeAg loss. Forty-eight weeks of ETV resulted in greater reductions in cccDNA and total hepatic HBV DNA than LAM, but long-term therapy may be needed for cccDNA elimination.04/2015; 21(15):4644-4651. DOI:10.3748/wjg.v21.i15.4644
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ABSTRACT: Chronic hepatitis B virus (HBV) infection is a leading cause of hepatitis, liver failure, and hepatocellular carcinoma. An outstanding vaccine is available; however the number of infections remains high. Current anti-HBV treatments with interferon α and nucleos(t)ide analogs clear the infection in only a small minority of patients, and either induce serious side-effects or are of very long duration. HBV is a small, enveloped DNA virus that replicates by reverse transcription via an RNA intermediate. The HBV ribonuclease H (RNaseH) is essential for viral replication, but it has not been exploited as a drug target. Recent low-throughput screening of compound classes with anti-Human Immunodeficiency Virus RNaseH activity led to identification of HBV RNaseH inhibitors in three different chemical families that block HBV replication. These inhibitors are promising candidates for development into new anti-HBV drugs. The RNaseH inhibitors may help improve treatment efficacy enough to clear the virus from the liver when used in combination with existing anti-HBV drugs and/or with other novel inhibitors under development. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B." Copyright © 2015. Published by Elsevier B.V.Antiviral research 04/2015; 118. DOI:10.1016/j.antiviral.2015.04.002 · 3.43 Impact Factor