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HBV RNaseH inhibitors: Effects on HBV biology and resistance development

HBV RNaseH 抑制剂：对 HBV 生物学和耐药性发展的影响

基本信息

批准号：
10064128
负责人：
JOHN E TAVIS
金额：
$ 37.88万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-02 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10064128
关键词：
Address Affect Antiviral Agents Biological Assay Biology Capsid Cell Culture Techniques Cells Chronic Circular DNA Clinical Collection Communication Complex DNA DNA Viruses DNA biosynthesis DNA-Directed DNA Polymerase Data Development Disease Progression Drug Targeting Endoplasmic Reticulum Enzymes Genome Hepatitis B Hepatitis B Therapy Hepatitis B Virus Hepatitis B virus Pol Protein Infection Interferons Kinetics Life Link Liver Failure Malignant neoplasm of liver Mutation Nuclear Patients Pharmaceutical Preparations Play Polymerase Protein Isoforms Proteins RNA RNA-Directed DNA Polymerase Recombinants Recycling Resistance Resistance development Reticulum Reverse Transcription Ribonuclease H Role Site Time Translations Viral Viral Genome Virion Virus Virus Integration Virus Replication Work analog detection limit drug discovery fitness genetic regulatory protein improved inhibitor/antagonist monomer novel therapeutics resistance mutation response screening tool tumorigenesis viral DNA viral RNA viral fitness

项目摘要

Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects about 290 million people and kills ~900,000 annually. Therapy primarily employs nucleos(t)ide analogs that target the viral DNA polymerase (P), but only a few percent of patients clear the virus so therapy is life-long. Reverse transcription is catalyzed by coordinate action of the viral DNA polymerase (RT) that synthesized the DNA and the ribonuclease H (RNaseH) that destroys the RNA after it has been copied into DNA. The RT and RNaseH comprise adjacent domains of P, and the 2 enzymatic activities are allosteric and/or kinetically linked during replication. Newly synthesized HBV genomes have 3 fates: to become nuclear covalently closed circular DNA (cccDNA) molecules, be secreted within virions, or be integrated into the cellular genome. P is also a regulatory protein that accumulates at the endoplasmic reticulum and helps suppress interferon responses. The role of the RT in HBV replication is fairly well understood, but very little is known about how the RNaseH contributes to HBV biology. We recently expressed recombinant HBV RNaseH suitable for mechanistic analyses and drug discovery for the first time. We then developed the first screening pipeline for HBV RNaseH inhibitors and identified >130 compounds that block HBV replication by inhibiting the RNaseH. The recombinant RNaseH and inhibitors are unique new tools to probe contributions of the RNaseH to viral biology. Premise: Our recombinant HBV RNaseH, RNaseH inhibitors, and RNaseH assays enable studies to reveal how the RNaseH contributes to viral biology and how the enzyme can evolve resistance to RNaseH inhibitors. Aim 1. What are the effects of inhibiting the RNaseH on HBV reverse transcription? We will evaluate how RNaseH inhibitors affect RNA encapsidation, the fate of the RNA during reverse transcription when the RNaseH is inhibited, reversibility of damage to the viral genome induced by RNaseH inhibitors, how blocking the RNaseH affects cccDNA synthesis, and the specific infectivity of virions made without RNaseH activity. Aim 2. What is the potential for resistance to RNaseH inhibitors? We will select resistance mutations to RNaseH inhibitors in cell culture and then define their effects on viral fitness and selectivity against inhibitors from 3 chemotypes. Aim 3. How does inhibiting the RNaseH affect capsids and HBV’s interaction with cells? We will define how RNA:DNA heteroduplexes generated by inhibiting the RNaseH affect HBV capsids, the effects of RNA:DNA heteroduplexes induced by RNaseH inhibitors on interferon responses, and how heteroduplexes made without RNaseH activity affect integration of HBV DNA into the host genome. These data will define how the HBV RNaseH contributes to viral biology. This information will deepen our understanding of HBV’s interaction with cells and provide essential context for our ongoing development of RNaseH inhibitors as novel drugs intended to improve therapy for chronically infected patients.

B型肝炎病毒（HBV）是一种嗜肝DNA病毒，通过逆转录进行复制。它会慢性感染约2.9亿人，每年杀死约90万人。治疗主要采用靶向的核苷（酸）类似物，病毒DNA聚合酶（P），但只有百分之几的患者清除病毒，所以治疗是终身的。反向转录由合成DNA的病毒DNA聚合酶（RT）的协同作用催化，核糖核酸酶H（RNaseH），在RNA复制成DNA后将其破坏。RT和RNaseH 包括P的相邻结构域，并且2种酶活性在复制的新合成的HBV基因组有三种命运：成为核共价闭合的环状DNA （cccDNA）分子，在病毒粒子内分泌，或整合到细胞基因组中。P也是一种监管在内质网中积累并帮助抑制干扰素反应的蛋白质。 RT在HBV复制中的作用已相当清楚，但关于RNaseH如何在HBV复制过程中起作用却知之甚少。有助于HBV生物学。我们最近表达了适合于机制分析的重组HBV RNaseH 和药物发现的机会。然后，我们开发了HBV RNaseH抑制剂的第一个筛选管道并鉴定了>130种通过抑制RNaseH阻断HBV复制的化合物。重组RNaseH 和抑制剂是探测RNaseH对病毒生物学的贡献的独特的新工具。前提：我们的重组HBV RNaseH、RNaseH抑制剂和RNaseH检测能够通过研究揭示 RNaseH如何促进病毒生物学以及该酶如何进化出对RNaseH抑制剂的抗性。目标1.抑制RNA酶H对HBV逆转录有什么影响？我们将评估如何 RNaseH抑制剂影响RNA的转录，即当RNaseH抑制RNA时，RNA在逆转录过程中的命运。是抑制，可逆性的损害病毒基因组诱导的RNaseH抑制剂，如何阻断RNaseH 影响cccDNA的合成，以及无RNaseH活性的病毒体的特异性感染性。目标2.对RNaseH抑制剂产生耐药性的可能性是什么？我们将选择耐药突变， RNaseH抑制剂在细胞培养中的作用，然后确定它们对病毒适应性和对抑制剂的选择性的影响三种化学类型目标3.抑制RNaseH如何影响衣壳和HBV与细胞的相互作用？我们将定义通过抑制RNaseH产生的RNA：DNA异源双链体如何影响HBV衣壳，RNA：DNA的影响 RNaseH抑制剂诱导的异源双链体对干扰素应答的影响，以及在没有RNaseH抑制剂的情况下如何产生异源双链体 RNaseH活性影响HBV DNA整合入宿主基因组。这些数据将定义HBV RNaseH如何促进病毒生物学。这些信息将加深我们的了解HBV与细胞的相互作用，并为我们正在进行的开发提供必要的背景， RNaseH抑制剂作为旨在改善慢性感染患者治疗的新药。