Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA.
PLoS Pathog. 2013 Jan;9(1):e1003125. doi: 10.1371/journal.ppat.1003125. Epub 2013 Jan 22.
Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 µM, the best compounds had low micromolar IC(50) values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 µM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development.
核苷酸类似物治疗通过乙型肝炎病毒（HBV）逆转录酶阻断 DNA 合成，能够控制感染，但治疗是终身的，成本高，且长期效果不可预测。核苷酸类似物对 HBV 的深度抑制作用以及它们能够治愈一些患者的能力表明，它们可以使 HBV 濒临灭绝。因此，通过使用新药物与核苷酸类似物联合进一步抑制 HBV 复制，更多的患者可以被治愈。HBV 核糖核酸酶 H（RNAseH）是一个合理的药物靶点，因为它是仅有的两种对病毒复制至关重要的病毒酶中的第二种，但尚未被利用，主要是因为它很难产生有活性的酶。为了解决这个困难，我们在大肠杆菌中表达了 HBV 基因型 D 和 H RNAseHs，并通过镍亲和力层析对酶进行了富集。在准备药物筛选之前，对富集的裂解物中的 HBV RNAseH 活性进行了表征。通过基于 HIV RNAseH 和整合酶抑制剂的化学结构-活性分析，鉴定了 21 种候选 HBV RNAseH 抑制剂。12 种抗 RNAseH 和抗整合酶化合物在 10µM 时抑制 HBV RNAseH，最佳化合物对 RNAseH 的 IC50 值较低，有 1 种化合物在 10µM 时抑制组织培养中的 HBV 复制。重组 HBV 基因型 D RNAseH 比基因型 H 更敏感。本研究证明已生产出适合低通量抗病毒药物筛选的重组 HBV RNAseH。针对 HIV RNAseH 和整合酶开发的化合物中有很高比例对 HBV RNAseH 有活性，这表明针对这些 HIV 酶的广泛药物设计工作可以指导抗 HBV RNAseH 药物发现。最后，HBV 基因型 D 和 H RNAseHs 的差异抑制表明病毒遗传变异性将是药物开发过程中的一个因素。
