Development of an HTS Assay for Discovery of HBV cccDNA Inhibitors

开发用于发现 HBV cccDNA 抑制剂的 HTS 测定法

• 批准号: 9236941
• 负责人: Haitao Guo
• 金额: $ 40.47万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9236941
• 关键词: Alpha Cell; Antibodies; Antigens; Antiviral Agents; Biological Assay; Cell Culture System; Cell Culture Techniques; Cell Line; Cells; Characteristics; Chemicals; Chronic; Chronic Hepatitis B; Circular DNA; Code; Collaborations; Collection; Complex; Coupled; Cross Reactions; DNA Maintenance; DNA biosynthesis; DNA-Directed DNA Polymerase; Dependency; Derivation procedure; Detection; Development; Disease; Dose; Elements; Enzymes; Epitopes; Excision; Exhibits; FDA approved; Future; Generations; Genetic Transcription; Genome; Genomics; Goals; Hand; Hepatitis B; Hepatitis B Core Antigen; Hepatitis B e Antigens; Hepatocyte; Homologous Gene; Immunology procedure; Infection; Lead; Libraries; Life Cycle Stages; Link; Longevity; Maintenance; Measurable; Measurement; Measures; Messenger RNA; N-terminal; Noise; Nucleic Acids; Open Reading Frames; Patients; Performance; Pharmaceutical Preparations; Polymerase; Production; Proteins; RNA; Reporter; Repression; Reproducibility; Research; Sensitivity and Specificity; Signal Transduction; Specificity; Structure-Activity Relationship; System; Testing; Therapeutic; Transcript; Transgenes; Universities; Validation; Viral; Viral Genome; Virus; Virus Diseases; Virus Inhibitors; Virus Replication; analog; anti-hepatitis B; assay development; base; cytotoxicity; cytotoxicity test; high throughput screening; improved; inhibitor/antagonist; miniaturize; novel; prototype; reconstitution; response; screening; small molecule; small molecule libraries; stable cell line; targeted treatment; tissue culture; tool; viral DNA

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.

摘要 本研究旨在建立一种新型的高通量检测B型肝炎抑制剂的方法 病毒（HBV）共价闭合环状DNA（cccDNA）。HBV cccDNA对病毒生命周期至关重要， 慢性感染期间的消除被认为是持久治疗的关键，但尚未实现， FDA批准了专门针对病毒聚合酶的小分子抗病毒药物。但由于 目前HBV组织培养系统的局限性，包括检测cccDNA本身的不切实际性， cccDNA在小分子文库的高通量筛选（HTS）中尚未被严格靶向。在这 建议，一种新的组织培养系，表达HA表位标记的B e肝炎抗原（HBeAg）， cccDNA依赖性方式将用于开发用于发现cccDNA抑制剂的基于细胞的HTS测定。 该细胞系从稳定整合的HBV基因组（转基因）诱导产生病毒前基因组转录物 在前核心区插入HA表位序列，导致病毒DNA基因组复制， cccDNA形成;随后，HA标记的HBeAg RNA和蛋白质仅由转录物制成 当从cccDNA模板产生HA-HBeAg时，HA-HBeAg分泌到细胞培养上清液中。的 HA标签掺入HBeAg是为了避免在基于ELISA的检测中HBeAg与HBcAg的交叉反应。 免疫学测定，如化学发光ELISA和AlphaLISA。在HTS活动中，化合物 降低HA-HBeAg将被认为是cccDNA形成、表达或 中心blog通过与Purdue Chemical Genomics Facility的专业HTS团队合作 （PCGF），我们将首先将抗病毒测定法应用于384孔格式，并将其性能特征和 将确定HTS条件（包括Z '）下试验的耐用性。接下来，将显示一个试点屏幕 针对含有10，560个“精选”化合物的PCGF子库进行，第一轮命中将是 通过剂量范围活性和细胞毒性分析以及通过细胞系中的反筛选进行过滤 组成型表达转基因（非cccDNA）依赖性HA-HBeAg以去除脱靶命中。最后， 通过直接测量以下物质的水平，在产生cccDNA的细胞系中评价HTS衍生的命中物： cccDNA和/或其转录物。确认的命中和他们的类似物也将重新合成和重新测试， 其针对cccDNA的活性以获得最终命中。因此，该项目的完成将提供一个 HTS平台用于从大型化合物库中发现新型HBV抑制剂;中试筛选和命中 验证工作将扩大用于HBV研究的化合物库，甚至可能 慢性B型肝炎转化疗法的衍生。