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

描述（由申请人提供）：乙型肝炎病毒（HBV）是一种通过逆转录复制的嗜肝性DNA病毒。它长期感染全球3.5亿人，每年导致120万患者死亡。治疗方法采用核苷类似物，可以很好地抑制病毒DNA合成，但即使经过多年的治疗，它们也只能治愈百分之几的患者。重要的是，核苷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。这为进一步抑制乙肝病毒治愈许多患者提供了巨大的机会。所有HBV DNA都是在细胞质中通过2种病毒酶活性的作用，即合成DNA的DNA聚合酶和在病毒RNA被复制成DNA后破坏病毒RNA的核糖核酸酶H (RNAse H)，通过逆转录产生的。新合成的基因组既可以被包膜并以病毒粒子的形式从细胞中分泌出来，也可以被运输到细胞核中以补充病毒基因组的外体形式（“cccDNA”）。cccDNA是维持HBV感染的关键，因为它是HBV转录的模板，而治疗HBV意味着消除cccDNA。在血清中病毒滴度被核苷类似物抑制到临床检测极限以下的患者中，cccDNA的无限期持续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核cccDNA库。因此，迫切需要能够作用于DNA聚合酶活性位点以外靶点的新型药物。这些药物将与现有的核苷类似物一起使用，以抑制HBV复制，使其低于维持cccDNA所需的水平。HBV RNAse H活性一直不是药物开发的焦点，尽管它是一个有吸引力的靶标，因为溶解度和稳定性问题阻碍了适合药物筛选的活性RNAse H的产生。我们现在可以生产活性重组HBV RNAse 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RNAse H抑制剂的高通量筛选（HTS），为抗HBV RNAse H药物开发做准备。该HTS将与我们现有的生化和基于细胞的RNAse H检测相结合，为开发新型抗hbv RNAse H药物提供整合的生化到细胞培养管道。目的1。HTS分析开发和性能验证。我们的RNAse H测定将适应荧光格式，并基于先前用于鉴定抗hiv RNAse H化合物的类似测定进行小型化。建立该方法在高温高温条件下的性能特征。目标2。抗hbv RNAse H化合物的原理证明HTS。将筛选6588种化合物，以证明该试验的稳健性，并指导文库选择，以进行未来的药物筛选。

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.