Development of an HTS Assay for Discovery of HBV cccDNA Inhibitors

摘要 这是一项建议，以开发一种新的高通量检测系统来检测乙肝抑制物 病毒(HBV)共价闭合环状DNA(CccDNA)。HBVcccDNA对病毒的生命周期是必不可少的，其 慢性感染期间的消除被认为是持久治疗的关键，但尚未通过 FDA批准了专门针对病毒聚合酶的小分子抗病毒药物。然而，由于 目前乙肝病毒组织培养系统的局限性，包括检测cccDNA本身的不切实际， 在小分子文库的高通量筛选(HTS)中，cccDNA还没有被严格地作为靶点。在这 一种新的表达HA表位标记的乙肝e抗原(HBeAg)的组织培养株 CccDNA依赖的方式将被用来建立一种基于细胞的HTS检测方法，用于发现cccDNA抑制剂。 该细胞系从稳定整合的乙肝病毒基因组(转基因)诱导产生病毒前基因组转录本。 在前核心区插入HA表位序列，导致病毒DNA基因组和 CccDNA的形成；随后，HA标记的HBeAg RNA和蛋白质只能从转录本中获得 从cccDNA模板中产生HA-HBeAg，然后分泌到细胞培养上清中。这个 在HBeAg中引入HA-Tag可避免HBeAb与HBcAg的交叉反应 免疫学检测：化学发光法、ELISA法、AlphaLISA法。在HTS活动中，化合物 降低HA-HBeAg可能被认为是cccDNA形成、表达或 长寿。通过与普渡化学基因组设施的专业HTS团队合作 (PCGF)，我们将首先将抗病毒检测小型化到384孔格式、性能特征和 将确定该分析在包括Z‘在内的HTS条件下的稳健性。接下来，将显示Pilot屏幕 对PCGF亚库进行的研究包含10,560种“精选”化合物，第一轮的命中结果将是 通过剂量范围活性和细胞毒性分析以及细胞系中的反筛选进行筛选 组成性表达依赖于转基因(而不是cccDNA)的HA-HBeAg，以去除靶外靶标。最后， HTS衍生的HITS将在产生cccDNA的细胞系中通过直接测量 CccDNA和/或其转录本。确认的命中和它们的类似物也将被重新合成和重新测试 它们对cccDNA的活性以获得最终的命中。因此，这个项目的完成将带来一个 HTS平台用于从较大的化合物库中发现新的乙肝病毒抑制剂；中试筛选和点击 验证工作将扩大用于乙肝研究的化合物池，甚至可能 慢性乙型病毒性肝炎转化疗法的派生

ABSTRACT This is a proposal to develop a novel high throughput assay system for detection of inhibitors of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). HBV cccDNA is essential to the virus life cycle, its elimination during chronic infection is considered critical to durable therapy but has not been achieved by the FDA approved small molecule antiviral drugs that exclusively target the viral polymerase. However, because of the limitations of current HBV tissue culture systems, including the impracticality of detecting cccDNA itself, cccDNA has not been rigorously targeted in high throughput screening (HTS) of small molecule libraries. In this proposal, a novel tissue culture line that expresses HA epitope-tagged hepatitis B e antigen (HBeAg) in a cccDNA-dependent manner will be used to develop a cell-based HTS assay for discovery of cccDNA inhibitors. This cell line inducibly produces viral pregenome transcripts from a stably integrated HBV genome (transgene) with an HA epitope sequence inserted in the precore region, leading to replication of viral DNA genome and cccDNA formation; subsequently, HA-tagged-HBeAg RNA and protein are only made from transcripts produced from the cccDNA template, then HA-HBeAg is secreted into the cell culture supernatant. The incorporation of HA-tag into HBeAg is to avoid the cross reaction of HBeAb with HBcAg in the ELISA-based immunological assays, such as chemiluminescence ELISA and AlphaLISA. In an HTS campaign, compounds that lower the HA-HBeAg would be considered candidate inhibitors of cccDNA formation, expression or longevity. Through collaboration with the professional HTS team in the Purdue Chemical Genomics Facility (PCGF), we will first miniaturize the antiviral assay to the 384-well format, the performance characteristics and the robustness of the assay under HTS conditions, including Z’, will be determined. Next, a pilot screen will be conducted against a PCGF sublibrary containing 10,560 “cherry-picked” compounds, the first round hits will be filtered through dose-ranging activity and cytotoxicity analyses, and through counter-screening in a cell line constitutively expressing transgene- (not cccDNA-) dependent HA-HBeAg to remove the off-target hits. Finally, the HTS-derived hits will be evaluated in cccDNA-producing cell lines by directly measuring the levels of cccDNA and/or its transcripts. The confirmed hits and their analogs will also be resynthesized and retested for their activity against cccDNA to obtain the final hits. Thus, the accomplishment of this project will deliver an HTS platform for discovery of novel HBV inhibitors from larger compound libraries; the pilot screen and hit validation effort will expand the pool of compounds to be used for HBV research, or even the possible derivation of transformational therapies for chronic hepatitis B.