Identification of Disubstituted Sulfonamide Compounds as Specific Inhibitors of Hepatitis B Virus Covalently Closed Circular DNA Formation
Identification of Disubstituted Sulfonamide Compounds as Specific Inhibitors of Hepatitis B Virus Covalently Closed Circular DNA Formation
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DOI：
10.1128/aac.00473-12
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发表时间：
2012-08-01
影响因子：
4.9
通讯作者：
Guo, Haitao
中科院分区：
文献类型：
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作者：
Cai, Dawei;Mills, Courtney;Guo, Haitao
B型肝炎病毒（HBV）共价闭合环状DNA（cccDNA）在病毒感染和持续性中起着核心作用，是治疗停止后病毒反弹的基础，也是延长治疗后治愈的不确定性的基础。因此，迫切需要开发直接靶向cccDNA形成和维持的新型治疗剂。通过采用一种创新的基于细胞的cccDNA测定法，其中分泌的HBV e抗原是cccDNA依赖性替代物，我们筛选了由85，000种药物样化合物组成的内部小分子文库。出现了两种结构相关的二取代磺酰胺（DSS），称为CCC-0975和CCC-0346，并被确认为cccDNA产生的抑制剂，在细胞培养物中具有低微摩尔50%有效浓度（EC（50）s）。进一步的机制研究表明，DSS化合物处理既不直接抑制细胞培养物中的HBV DNA复制，也不降低体外内源性聚合酶测定中的病毒聚合酶活性，但同步降低HBV cccDNA及其推定前体脱蛋白松弛环状DNA（DP-rcDNA）的水平。然而，DSS化合物不促进HBV DP-rcDNA和cccDNA的细胞内衰变，表明化合物主要干扰rcDNA转化为cccDNA。此外，我们证明CCC-0975能够减少鸭HBV感染的原代鸭肝细胞中cccDNA的生物合成。据我们所知，这是鉴定靶向cccDNA形成的小分子的第一次尝试，因此DSS化合物可能作为概念验证药物候选物，用于开发治疗剂以消除慢性HBV感染中的cccDNA。
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.