Intervention of HBV DNA synthesis and transcription

干预 HBV DNA 合成和转录

• 批准号: 8511268
• 负责人: ALEEM SIDDIQUI
• 金额: $ 21.86万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-06 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8511268
• 关键词: 9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine; Address; Affect; Anti-HIV Agents; Antiviral Agents; Antiviral Therapy; Bioavailable; Biological Assay; Cell Line; Cell Nucleus; Cells; Chronic; Chronic Hepatitis B; Cidofovir; Circular DNA; Cytomegalovirus; DNA; DNA Structure; DNA Viruses; DNA biosynthesis; Development; Ectromelia; Equus caballus; Esters; Exhibits; Fibrosis; Genetic Transcription; Genome; Genomics; Goals; Grant; HIV; Hepatitis B Virus; Hepatocyte; In Vitro; Incubated; Infection; Intervention; Label; Lead; Lipids; Maintenance; Measures; Mediating; Messenger RNA; Molecular; Molecular Biology; Nuclear; Nucleocapsid; Nucleosides; Nucleosome Core Particle; Nucleotides; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Polymerase; Predisposition; Primary carcinoma of the liver cells; Procedures; Production; Protein Biosynthesis; Proteins; RNA; RNA-Directed DNA Polymerase; Radioactive; Radiolabeled; Reaction; Recycling; Refractory; Relapse; Relative (related person); Reporting; Reverse Transcription; Risk; Risk Factors; Smallpox; Solutions; Structure; Testing; Tetracyclines; Time; Toxic effect; Transgenic Mice; Vaccinia; Vaccinium; Viral; Virion; Virus; Virus Diseases; Virus Replication; Withdrawal; Work; adefovir dipivoxil; analog; anti-hepatitis B; base; chemotherapy; design; ds-DNA; hepatitis B virus P protein; in vivo; innovation; novel; nucleoside analog; phosphonate; pol Gene Products; prevent; promoter; public health relevance; radiotracer; treatment duration; uptake; viral DNA; viral RNA

DESCRIPTION (provided by applicant): Chronic hepatitis B virus infections affect about 300 million people worldwide and constitute a significant risk factor for fibrosis and hepatocellular carcinoma (HCC). HBV, a DNA virus, amplifies its genome via a pre-genomic RNA. This RNA is converted by HBV-encoded polymerase protein (pol), which functions as a reverse transcriptase, into DNA. The final product of this reaction is a partially double stranded DNA, which is converted to covalently closed circular DNA (cccDNA) in the nucleus cccDNA serves as a template for viral mRNA synthesis and proteins. cccDNA persists in the presence of the antivirals in current use, which act as chain terminators. We (Hostetler and colleagues) have previously shown that the acyclic nucleoside phosphonates, HPMPC and HPMPA are incorporated into viral DNA. Viral DNA templates containing these bases cannot be copied readily and have aberrations in DNA structure. In this study, we propose to investigate the incorporation of these agents into HBV DNA during the conversion from the RNA pregenome within the core particles and subsequent synthesis of cccDNA. In the R21 phase of the grant, we investigate the incorporation of radiolabeled HPMPA/HPMPC and its impact by analysis of viral DNA within core particles and finally the formation of cccDNA. Effect of these drugs on viral mRNA and protein synthesis will be analyzed to test the template activity of cccDNA containing HPMPA/HPMPC. In the second R33 phase, we propose to define the changes in duplex DNA containing these nucleotides. We will synthesize several templates containing HPMPA or HPMPC and assess the NMR solution structures of their duplex DNA versus control DNA duplexes. Aim 4 of R33 phase explores the impact of these drugs in the context of HBV infection using a cell line HepaRG that is widely used in the HBV field and recognized for its susceptibility to HBV infection. These studies will provide a unique opportunity to assess the potential new way to disable and ultimately eliminate the HBV cccDNA and prevent its expression. Our hypothesis is that incorporation of HMPMA into the DNA induces structural abnormalities, and in this case possibly destabilizing cccDNA, altering its structure and leading to its elimination over time. Our team combines the state of the art medicinal chemistry (Hostetler) and HBV molecular biology (Siddiqui) expertise to address this long standing problem of HBV viral persistence of cccDNA in chronic infection and being refractory to antiviral strategies currently in vogue.

描述（由申请人提供）：慢性乙型肝炎病毒感染影响全球约 3 亿人，并构成纤维化和肝细胞癌 (HCC) 的重要危险因素。 HBV 是一种 DNA 病毒，通过前基因组 RNA 扩增其基因组。这种 RNA 被 HBV 编码的聚合酶蛋白 (pol)（充当逆转录酶）转化为 DNA。该反应的最终产物是部分双链 DNA，它在细胞核中转化为共价闭合环状 DNA (cccDNA)，cccDNA 用作病毒 mRNA 合成和蛋白质的模板。 cccDNA 在当前使用的抗病毒药物存在的情况下持续存在，抗病毒药物充当链终止剂。我们（Hostetler 和同事）之前已经证明，无环核苷膦酸酯、HPMPC 和 HPMPA 被整合到病毒 DNA 中。含有这些碱基的病毒 DNA 模板不易复制，并且 DNA 结构存在畸变。在本研究中，我们建议研究在核心颗粒内的 RNA 前基因组转化以及随后的 cccDNA 合成过程中这些试剂掺入 HBV DNA 的情况。在资助的 R21 阶段，我们通过分析核心颗粒内的病毒 DNA 以及最终 cccDNA 的形成来研究放射性标记 HPMPA/HPMPC 的掺入及其影响。这些药物对病毒的作用 将分析 mRNA 和蛋白质合成，以测试含有 HPMPA/HPMPC 的 cccDNA 的模板活性。在第二个 R33 阶段，我们建议定义包含这些核苷酸的双链 DNA 的变化。我们将合成几个含有 HPMPA 或 HPMPC 的模板，并评估其双链体 DNA 与对照 DNA 双链体的 NMR 溶液结构。 R33 阶段的目标 4 使用细胞系 HepaRG 探讨这些药物在 HBV 感染情况下的影响，HepaRG 细胞系广泛用于 HBV 领域，并因其对 HBV 感染的易感性而受到认可。这些研究将提供一个独特的机会来评估禁用并最终消除 HBV cccDNA 并阻止其表达的潜在新方法。我们的假设是，HMPMA 掺入 DNA 会引起结构异常，在这种情况下可能会破坏 cccDNA 的稳定性，改变其结构并导致其随着时间的推移而被消除。我们的团队结合了最先进的药物化学 (Hostetler) 和 HBV 分子生物学 (Siddiqui) 专业知识，以解决慢性感染中 cccDNA 的 HBV 病毒持续存在以及当前流行的抗病毒策略难以治愈的长期存在的问题。