新型乙型肝炎病毒基因组RNA衣壳化抑制剂的鉴定和表征

StephenW

Antiviral Res. 2020 Jan 12:104709. doi: 10.1016/j.antiviral.2020.104709. [Epub ahead of print]
Identification and characterization of a novel hepatitis B virus pregenomic RNA encapsidation inhibitor.
Jo E1, Ryu DK1, König A1, Park S2, Cho Y1, Park SH1, Kim TH2, Yoon SK3, Ryu WS4, Cechetto J2, Windisch MP5.
Author information

1
    Applied Molecular Virology Laboratory, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea.
2
    Screening Discovery Platform, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea.
3
    Catholic University Liver Research Center, The Catholic University of Korea, Seoul, South Korea.
4
    Department of Biochemistry, Yonsei University, Seoul, South Korea.
5
    Applied Molecular Virology Laboratory, Institut Pasteur Korea, 696 Sampyung-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea; Division of Bio-Medical Science and Technology, University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Daejeon, South Korea. Electronic address: [email protected].

Abstract

Currently, therapies to treat chronic hepatitis B (CHB) infection are based on the use of interferon-α or nucleos(t)ide analogs (NAs) to prevent viral DNA synthesis by inhibiting the reverse transcriptase activity of the hepatitis B virus (HBV) polymerase (Pol). However, these therapies are not curative; thus, the development of novel anti-HBV agents is needed. In accordance with this unmet medical need, we devised a new target- and cell-based, high-throughput screening assay to identify novel small molecules that block the initial interaction of the HBV Pol with its replication template the viral pregenomic RNA (pgRNA). We screened approximately 110,000 small molecules for the ability to prevent HBV Pol recognition of the pgRNA 5' epsilon (ε) stem-loop structure, identifying (Z)-2-(allylamino)-4-amino-N'-cyanothiazole-5-carboximidamide (AACC). Viral nucleocapsid-captured quantitative RT-PCR and Western blot results revealed that AACC significantly decreased encapsidated pgRNA levels and blocked capsid assembly without affecting core protein expression in stable HBV-replicating cells. As a result, both intra- and extracellular accumulation of viral DNA was strongly reduced. AACC treatment of HepG2-sodium taurocholate transporting polypeptide (NTCP) cells and primary human hepatocytes infected with cell culture- or patient-derived HBV isolates showed both time- and dose-dependent inhibition of infectious viral progeny and rcDNA production. Furthermore, AACC showed cross-genotypic activity against genotypes B, C, and D. Of note, AACC inhibited the viral replication of lamivudine and a capsid inhibitor-resistant HBV, and showed synergistic effects with NAs and a capsid inhibitor. In conclusion, we identified a novel class of compounds specifically targeting the ε-Pol interaction and thereby preventing the encapsidation of pgRNAs into viral capsids. This promising new HBV inhibitor class potently inhibits HBV amplification with distinct characteristics from existing NAs and other drugs currently under development, promising to add value to existing therapies for CHB.

Copyright © 2020. Published by Elsevier B.V.
KEYWORDS:

Encapsidation; Epsilon signal; Hepatitis B virus; High-throughput drug screening; Polymerase; Pregenomic RNA

PMID:
    31940474
DOI:
    10.1016/j.antiviral.2020.104709

StephenW

抗病毒水库。 2020年1月12日：104709。Doi：10.1016 / j.antiviral.2020.104709。 [Epub提前发行]
新型乙型肝炎病毒基因组RNA衣壳化抑制剂的鉴定和表征。
Jo E1，Ryu DK1，KönigA1，Park S2，Cho Y1，Park SH1，Kim TH2，Yoon SK3，Ryu WS4，Cechetto J2，Windisch MP5。
作者信息

1个
巴斯德研究所应用分子病毒学实验室，韩国京畿道城南市盆唐区三平洞696。
2
筛选发现平台，巴斯德研究所，韩国京畿道城南市盆唐区三平洞696。
3
韩国天主教大学天主教大学肝脏研究中心，韩国首尔。
4
延世大学生物化学系，韩国首尔。
5
巴斯德研究所应用分子病毒学实验室，韩国京畿道城南市盆唐区三平洞696号；科学技术大学生物医学科学技术系，韩国大田市九成区加井路217。电子地址：[email protected]。

抽象

当前，用于治疗慢性乙型肝炎（CHB）感染的疗法基于使用干扰素-α或核苷酸（t）类似物（NAs）通过抑制乙型肝炎病毒（HBV）的逆转录酶活性来预防病毒DNA合成聚合酶（Pol）。然而，这些疗法不是治愈性的。因此，需要开发新型抗HBV药物。根据未满足的医疗需求，我们设计了一种新的基于靶标和细胞的高通量筛选测定方法，以鉴定能够阻断HBV Pol及其复制模板与病毒前基因组RNA（pgRNA）初始相互作用的新型小分子。我们筛选了大约110,000个小分子，以防止其对pgRNA 5'ε（ε）茎-环结构进行HBV Pol识别，从而鉴定（Z）-2-（烯丙基氨基）-4-氨基-N'-氰基噻唑-5-羧酰亚胺（AACC）。病毒核衣壳捕获的定量RT-PCR和Western印迹结果表明，AACC显着降低了衣壳pgRNA的水平并阻止了衣壳组装，而没有影响稳定的HBV复制细胞中的核心蛋白表达。结果，病毒DNA在细胞内和细胞外的积累都大大减少了。 AACC处理的HepG2-牛磺胆酸钠运输多肽（NTCP）细胞和原代人肝细胞感染了细胞培养物或患者来源的HBV分离株，显示出感染病毒后代和rcDNA产生的时间和剂量依赖性抑制。此外，AACC对基因型B，C和D表现出交叉基因型活性。值得注意的是，AACC抑制了拉米夫定和衣壳抑制剂耐药的HBV的病毒复制，并显示了与NA和衣壳抑制剂的协同作用。总而言之，我们鉴定了一类新的化合物，这些化合物专门针对ε-Pol相互作用，从而防止pgRNA衣壳化为病毒衣壳。这种有前途的新型HBV抑制剂可有效抑制HBV扩增，具有与现有NAs和目前正在开发的其他药物不同的特征，有望为CHB的现有疗法增加价值。

版权所有©2020。由Elsevier B.V.发布。
关键字：

衣壳化； Epsilon信号；乙型肝炎病毒；高通量药物筛选；聚合酶；前基因组RNA

PMID：
31940474
DOI：
10.1016 / j.antiviral.2020.104709