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 这是一项开发用于检测乙型肝炎抑制剂的新型高通量测定系统的提案 病毒（HBV）共价闭合环状DNA（cccDNA）是HBV cccDNA对病毒生命周期至关重要的。 慢性感染期间的消除被认为对于持久治疗至关重要，但目前尚未实现 FDA 批准了专门针对病毒聚合酶的小分子抗病毒药物。 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， cccDNA 尚未成为小分子文库高通量筛选 (HTS) 的严格目标。 提议，一种新型组织培养系，可在细胞中表达 HA 表位标记的乙型肝炎 e 抗原 (HBeAg) cccDNA 依赖性方式将用于开发基于细胞的 HTS 测定，以发现 cccDNA 抑制剂。 该细胞系从稳定整合的 HBV 基因组（转基因）中诱导产生病毒前基因组转录本 在前核心区域插入 HA 表位序列，导致病毒 DNA 基因组的复制和 cccDNA 形成；随后，HA 标记的 HBeAg RNA 和蛋白质仅由转录物制成 从 cccDNA 模板产生，然后 HA-HBeAg 分泌到细胞培养上清液中。 将HA标签掺入HBeAg是为了避免基于ELISA的HBeAb与HBcAg的交叉反应 免疫学测定，例如化学发光 ELISA 和 AlphaLISA 在 HTS 活动中，化合物。 降低 HA-HBeAg 的药物将被视为 cccDNA 形成、表达或的候选抑制剂 通过与普渡化学基因组学设施的专业 HTS 团队合作。 (PCGF)，我们首先将抗病毒检测小型化至 384 孔格式，性能特征和 接下来，将确定该测定在 HTS 条件（包括 Z'）下的稳健性，并将进行试点筛选。 针对包含 10,560 个“精选”化合物的 PCGF 子库进行的测试，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 组成型表达转基因（非 cccDNA）依赖性 HA-HBeAg 以消除脱靶命中。 HTS 衍生的命中将通过直接测量 cccDNA 生产细胞系的水平进行评估 cccDNA 和/或其转录物也将被重新合成和重新测试。 他们针对 cccDNA 的活动以获得最终的结果，因此，该项目的完成将带来一个结果。 HTS 平台用于从更大的化合物库中发现新型 HBV 抑制剂； 验证工作将扩大用于 HBV 研究的化合物库，甚至可能 慢性乙型肝炎转化疗法的推导。