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.

丙型肝炎病毒（HBV）是一种通过逆转录复制的肝DNA病毒。它长期感染了大约2.9亿人，每年杀死约90万人。治疗初级雇员核心（T）IDE类似物病毒DNA聚合酶（P），但只有百分之几的患者清除病毒，因此治疗是终身的。撤销转录是通过合成DNA和DNA的病毒DNA聚合酶（RT）的坐标作用来催化的。核糖核酸酶H（RNASEH）在RNA被复制到DNA后破坏了RNA。 RT和RNASEH P的完整邻近域和2个酶促活性是变构和/或动力学链接的复制。新合成的HBV基因组具有3个命运：成为核共价闭合圆形DNA （CCCDNA）分子，分泌在病毒中，或集成到细胞基因组中。 P也是一个调节积聚在内质网状并有助于抑制干扰素反应的蛋白质。 RT在HBV复制中的作用相当理解，但对RNASEH的了解很少有助于HBV生物学。我们最近表达了适合机械分析的重组HBV RNASEH 和药物发现。然后，我们开发了HBV RNASEH抑制剂的第一个筛选管道并鉴定出> 130种通过抑制RNASEH来阻断HBV复制的化合物。重组rnaseh 抑制剂是探测RNASEH对病毒生物学的贡献的独特新工具。前提：我们的重组HBV RNASEH，RNASEH抑制剂和RNASEH分析使研究能够揭示 RNASEH如何有助于病毒生物学以及酶如何发展对RNAseH抑制剂的抗性。目标1。抑制RNASEH对HBV逆转录的影响是什么？我们将评估如何 RNASEH抑制剂会影响RNA封装，RNASEH时RNA逆转录过程中RNA的命运受到抑制，RNaseH抑制剂诱导的病毒基因组损害的可逆性如何阻止RNAseH 影响CCCDNA合成，并影响没有RNASEH活性的病毒的特定感染。目标2。抗RNASEH抑制剂的潜力是什么？我们将选择阻力突变 RNASEH抑制剂在细胞培养中，然后定义其对病毒适应性和针对抑制剂的影响的影响来自3个化学型。 AIM 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 RNA：通过抑制RNASEH产生的DNA杂化链影响HBV衣壳，RNA的作用：DNA RNASEH抑制剂在干扰素反应中诱导的杂化链，以及如何在没有的杂化响应中 RNASEH活性影响HBV DNA进入宿主基因组。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了解HBV与细胞的相互作用，并为我们的持续发展提供了基本背景 RNASEH抑制剂作为新型药物，旨在改善长期感染患者的治疗。