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.

抽象的 这是开发一种新型高通量测定系统以检测乙型肝炎抑制剂的建议 病毒（HBV）共价闭合圆形DNA（CCDNA）。 HBV CCCDNA对于病毒生命周期至关重要 慢性感染期间消除对耐用疗法至关重要，但尚未通过 FDA批准了仅针对病毒聚合酶的小分子抗病毒药物。但是，由于 当前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尚未在小分子文库的高吞吐量筛选（HTS）中进行严格针对。在这个 提案，一种新型的组织培养线，表达在A抗原抗原（HBEAG）中表达HA的组织培养线 CCCDNA依赖性方式将用于开发基于细胞的HTS测定法以发现CCCDNA抑制剂。 该细胞系可诱导地产生来自稳定集成的HBV基因组（Transgene）的病毒属前体转录本 插入前孔区域的HA表位序列，导致病毒DNA基因组的复制和 CCCDNA组；随后，标有HA的HBEAG RNA和蛋白质仅由转录本制成 由CCCDNA模板产生，然后将Ha-Hbeag分泌到细胞培养物上清液中。这 将HA-TAG掺入HBEAG是为了避免HBEAB与HBCAG的交叉反应 免疫学测定，例如化学发光ELISA和α。在HTS活动中，化合物 降低ha-hbeag将被视为CCCDNA形成，表达或 长寿。通过与普渡大学化学基因组学设施中的专业HTS团队合作 （PCGF），我们将首先将抗病毒测定法至384孔格式，性能特征和 在HTS条件（包括Z'）的HTS条件下，该测定的鲁棒性将被确定。接下来，试点屏幕将是 针对含有10,560个“挑选樱桃”化合物的PCGF巨纤维进行的，第一轮命中率是 通过剂量 - 含量活性和细胞毒性分析进行过滤，并在细胞系中进行反筛查 组成性表达转换 - （而非CCCDNA-）依赖性ha-hbeag去除脱离目标的命中。最后， HTS衍生的热门人士将通过直接测量的水平来评估CCCDNA产生的细胞系 CCCDNA和/或其成绩单。确认的命中及其类似物也将重新合成并重新测试 他们针对CCCDNA的活动以获得最终命中。那，这个项目的成就将提供 从较大的复合库中发现新型HBV抑制剂的HTS平台；飞行员屏幕并击中 验证工作将扩大用于HBV研究的化合物池，甚至可能 慢性肝炎的转化疗法的推导。