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

描述(申请人提供)：乙肝病毒(乙肝)是一种嗜肝的DNA病毒，通过逆转录进行复制。它在全球慢性感染3.5亿人，每年导致多达120万患者死亡。治疗使用核素(T)类似物，很好地抑制病毒DNA合成，但即使在多年的治疗后，它们也只治愈了几个百分点的患者。重要的是，核素类似物治愈一些患者的能力表明，它们可以将病毒推向消灭的边缘。这为通过进一步抑制乙肝病毒而治愈许多患者提供了一个巨大的机会。所有的HBVDNA都是通过两种病毒酶的作用在细胞质中反转录产生的，即合成DNA的DNA聚合酶和复制到DNA中后破坏病毒RNA的核糖核酸酶H(RNase H)。新合成的基因组可以被包裹并以病毒粒子的形式从细胞中分泌出来，或者它们可以被运输到细胞核中来补充病毒基因组的异构体形式(“cccDNA”)。CccDNA是维持乙肝病毒感染的关键，因为它是乙肝病毒转录的模板，而治愈乙肝意味着消除cccDNA。在血清病毒滴度被核素(T)类似物抑制到临床检测限度以下的患者中，cccDNA的无限期持续存在是残留病毒复制的结果。这导致通过细胞内基因组运输和新细胞的低水平感染来补充肝细胞核cccDNA池。因此，迫切需要作用于DNA聚合酶活性部位以外的靶点的新型药物。这些药物将与现有的核苷酸(T)类似物一起使用，以将乙肝病毒的复制抑制在维持cccDNA所需的水平以下。尽管HBVRNase H活性一直是一个有吸引力的靶点，但由于溶解性和稳定性问题阻碍了活性RNase H的生产，使其适用于药物筛选，因此一直不是药物开发的重点。我们现在可以生产活性的重组HBVRNase H。在这里，我们将与圣路易斯大学的专业药物发现团队密切合作，开发高通量RNase H抑制剂筛选(HTS)，为抗HBVRNase H药物的开发做准备。该HTS将与我们现有的生化和基于细胞的RNaseH分析相结合，为开发新型抗HBVRNase H药物提供一个从生物化学到细胞培养的集成管道。目的1.HTS检测方法的建立及性能验证。我们的RNaseH分析将被改造成荧光格式，并在以前用于鉴定抗HIV RNaseH化合物的类似分析的基础上进行小型化。将建立该分析在高温超导条件下的性能特征。目的2.抗乙肝核糖核酸酶H类化合物的原理验证。将对6588个化合物进行筛选，以证明该分析的稳健性，并指导未来药物筛选的文库选择。

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.