能够检测核糖核酸酶H抑制剂的中通量HBV复制抑制测定

StephenW

A mid-throughput HBV replication inhibition assay capable of detecting ribonuclease H inhibitors
Qilan Li  1 , Tiffany C Edwards  2 , Nathan L Ponzar  3 , John E Tavis  4
Affiliations
Affiliations

    1
    Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO, USA; Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO, USA. Electronic address: [email protected].
    2
    Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO, USA; Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO, USA. Electronic address: [email protected].
    3
    Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO, USA; Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO, USA. Electronic address: [email protected].
    4
    Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO, USA; Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO, USA. Electronic address: [email protected].

    PMID: 33766659 DOI: 10.1016/j.jviromet.2021.114127

Abstract

The hepatitis B virus (HBV) ribonuclease H (RNaseH) is a promising but unexploited drug target. Inhibiting the RNaseH blocks viral reverse transcription by truncating the minus-polarity DNA strand, causing accumulation of RNANA heteroduplexes, and abrogating plus-polarity DNA synthesis. Screening for RNaseH inhibitors is complicated by the presence of the minus-polarity DNA strand even when replication is fully inhibited because this residual DNA can be detected by standard screening assays that measure reduction in HBV DNA accumulation. We previously developed a strand-preferential qPCR assay that detects RNaseH replication inhibitors by measuring preferential suppression of the viral plus-polarity DNA strand. However, this assay employed cells grown in 6- or 12-well plates and hence was of very low throughput. Here, we adapted the assay to a 96-well format and conducted a proof-of-principle screen of 727 compounds. The newly developed assay is a valuable tool for anti-HBV drug discovery, particularly when screening for RNaseH inhibitors.

Keywords: Hepatitis B virus; assay optimization; replication inhibition assay; ribonuclease H.

Copyright © 2021. Published by Elsevier B.V.

StephenW

能够检测核糖核酸酶H抑制剂的中通量HBV复制抑制测定
李其兰1，蒂芙尼C爱德华兹2，内森·庞扎3，约翰·塔维斯4
隶属关系
隶属关系

    1个
    圣路易斯大学医学院分子微生物学和免疫学系，美国密苏里州圣路易斯；美国密苏里州圣路易斯市圣路易斯大学医学院圣路易斯大学肝脏中心。电子地址：[email protected]。
    2个
    圣路易斯大学医学院分子微生物学和免疫学系，美国密苏里州圣路易斯；美国密苏里州圣路易斯市圣路易斯大学医学院圣路易斯大学肝脏中心。电子地址：[email protected]。
    3
    圣路易斯大学医学院分子微生物学和免疫学系，美国密苏里州圣路易斯；美国密苏里州圣路易斯市圣路易斯大学医学院圣路易斯大学肝脏中心。电子地址：[email protected]。
    4
    圣路易斯大学医学院分子微生物学和免疫学系，美国密苏里州圣路易斯；美国密苏里州圣路易斯市圣路易斯大学医学院圣路易斯大学肝脏中心。电子地址：[email protected]。

    PMID：33766659 DOI：10.1016 / j.jviromet.2021.114127

抽象的

乙型肝炎病毒（HBV）核糖核酸酶H（RNaseH）是一种有前途但未开发的药物靶标。抑制RNaseH可以通过截断负极性DNA链，引起RNA：DNA异源双链体的积累以及废除正极性DNA的合成来阻断病毒逆转录。即使完全抑制了复制，由于存在负极性DNA链，使RNaseH抑制剂的筛选变得复杂，因为可以通过测量HBV DNA积累减少的标准筛选测定法检测到这种残留的DNA。我们先前开发了一种链优先qPCR分析法，该方法可通过测量对病毒加极性DNA链的优先抑制来检测RNaseH复制抑制剂。但是，该测定法使用在6孔或12孔板中生长的细胞，因此通量非常低。在这里，我们将分析方法调整为96孔格式，并进行了727种化合物的原理验证筛选。新开发的测定法是发现抗HBV药物的宝贵工具，尤其是在筛选RNaseH抑制剂时。

关键字：乙型肝炎病毒；分析优化；复制抑制试验核糖核酸酶H.

版权所有©2021，由Elsevier B.V.发布。