A screen for antiviral compounds targeting the Hepatitis B Virus ribonuclease H

筛选针对乙型肝炎病毒核糖核酸酶 H 的抗病毒化合物

基本信息

批准号：
8974218
负责人：
JOHN E TAVIS
金额：
$ 33.75万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-12-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8974218
关键词：
Active Sites Adverse effects Antiviral Agents Antiviral Therapy Biochemical Biochemistry Biological Assay Capsid Cell Culture Techniques Cell Nucleus Cells Characteristics Circular DNA Clinical Collaborations Collection Coupled Cytoplasm DNA DNA Viruses DNA biosynthesis DNA-Directed DNA Polymerase Detection Development Future Gel Genetic Transcription Genome Genomics Goals HIV Health Hepatic Hepatitis B Virus Infection Libraries Life Liver Maintenance Medicine Nuclear Pathway interactions Patients Performance Pharmaceutical Preparations Preclinical Drug Evaluation Preparation Production Protein Biosynthesis Recombinants Recycling Residual state Reverse Transcription Ribonuclease H Saints Serum Solubility Universities Validation Viral Viral Genome Viral Proteins Virion Virus Virus Diseases Virus Replication Work World Health analog anti-hepatitis B assay development base cost drug development drug discovery drug testing high throughput screening inhibitor/antagonist intrahepatic killings miniaturize novel novel therapeutics nucleoside analog particle prevent targeted treatment tool viral DNA viral RNA

项目摘要

DESCRIPTION (provided by applicant): Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects >350 million people worldwide and kills up to 1.2 million patients annually. Therapy employs nucleos(t)ide analogs that suppress viral DNA synthesis very well, but they cure only a few percent of patients even after years of treatment. Importantly, the ability of the nucleos(t)ide analogs to cure some patients indicates that they can push the virus to the brink of elimination. This presents a huge opportunity to cure many patients by suppressing HBV further. All HBV DNAs are produced by reverse transcription in the cytoplasm through the action of 2 viral enzymatic activities, the DNA polymerase that synthesizes the DNA and the ribonuclease H (RNAse H) that destroys the viral RNA after it has been copied into DNA. Newly synthesized genomes can either be enveloped and secreted from the cell as virions, or they can be transported into the nucleus to replenish the episomal form of the viral genome (the "cccDNA"). The cccDNA is key to maintenance of HBV infection because it is the template for HBV transcription, and curing HBV means eliminating the cccDNA. The indefinite persistence of the cccDNA in patients whose viral titres in serum have been suppressed below the limit of clinical detection by the nucleos(t)ide analogs is the result of residual viral replication. This leads to replenishment of the hepatic nuclear cccDNA pool by intracellular genomic transport and low-level infection of new cells. Therefore, novel drugs that act on targets other than the DNA polymerase active site are urgently needed. These drugs would be used together with the existing nucleos(t)ide analogs to suppress HBV replication below the level needed to maintain the cccDNA. The HBV RNAse H activity has not been a focus of drug development despite being an attractive target because solubility and stability problems prevented production of active RNAse H suitable for drug screening. We can now produce active recombinant HBV RNAse H. Here, we will work closely with the professional drug discovery team at Saint Louis University to develop a high-throughput screen (HTS) for RNAse H inhibitors in preparation for anti-HBV RNAse H drug development. This HTS will be coupled with our existing biochemical and cell-based RNAse H assays to provide an integrated biochemistry-to-cell culture pipeline for development of novel anti-HBV RNAse H drugs. Aim 1. HTS assay development and performance validation. Our RNAse H assay will be adapted to a fluorescent format and miniaturized based on similar assays previously used to identify anti-HIV RNAse H compounds. The performance characteristics of the assay under HTS conditions will be established. Aim 2. Proof-of-principle HTS for anti-HBV RNAse H compounds. 6588 compounds will be screened to demonstrate robustness of the assay and to guide library selection for future drug screening.

描述（由申请人提供）：丙型肝炎病毒（HBV）是一种通过反转录复制的肝DNA病毒。它长期感染了全球范围> 3.5亿人的3.5亿人，每年杀死多达120万患者。治疗采用核（T）IDE类似物，可以很好地抑制病毒DNA合成，但即使经过多年的治疗，它们也只能治愈几个患者。重要的是，核（T）IDE类似物治愈某些患者的能力表明他们可以将病毒推向消除的边缘。这为通过进一步抑制HBV的抑制提供了巨大的机会来治愈许多患者。所有HBV DNA都是通过2种病毒酶活性的作用在细胞质中的逆转录来产生的，这是合成DNA和核糖核酸酶H（RNase H）的DNA聚合酶，该聚合酶已将病毒RNA摧毁了病毒RNA后将其复制到DNA中。新合成的基因组可以作为病毒体从细胞中包裹并分泌，也可以将其转运到细胞核中以补充病毒基因组的偶发形式（“ CCCDNA”）。 CCCDNA是维持HBV感染的关键，因为它是HBV转录的模板，并且治愈HBV意味着消除CCCDNA。 CCCDNA在血清中的病毒滴度已被核（T）IDE类似物抑制临床检测极限的患者中无限期的持久性是残留病毒复制的结果。这导致通过细胞内基因组转运和新细胞的低水平感染来补充肝核CCCDNA池。因此，迫切需要对DNA聚合酶活性位点以外的其他靶标的新型药物。这些药物将与现有核（T）IDE类似物一起使用，以抑制低于维持CCCDNA所需水平的HBV复制。尽管溶解度和稳定性问题阻止了适用于药物筛查的活性RNase H，但HBV RNase H活性并不是药物开发的重点。现在，我们可以生产活跃的重组HBV RNaseH。在这里，我们将与圣路易斯大学的专业药物发现团队紧密合作，为RNase H抑制剂开发高通量筛查（HTS），以准备抗HBV RNase H酶H药物开发。该HTS将与我们现有的生物化学和基于细胞的RNase H分析相结合，为新型抗HBV RNase H药物的开发提供综合的生物化学到细胞培养管道。目标1。HTS测定开发和性能验证。我们的RNase H测定法将适用于荧光格式，并根据先前用于鉴定抗HIV RNase H化合物的类似测定法进行微型化。将在HTS条件下建立测定的性能特征。 AIM 2。抗HBV RNase H化合物的原则HTS。将筛选6588种化合物，以证明测定法的鲁棒性，并指导图书馆选择以进行未来的药物筛查。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Inhibition of hepatitis B virus replication by N-hydroxyisoquinolinediones and related polyoxygenated heterocycles.

DOI：
10.1016/j.antiviral.2017.04.012
发表时间：
2017-07
期刊：
Antiviral research
影响因子：
7.6
作者：
Edwards TC;Lomonosova E;Patel JA;Li Q;Villa JA;Gupta AK;Morrison LA;Bailly F;Cotelle P;Giannakopoulou E;Zoidis G;Tavis JE
通讯作者：
Tavis JE