开发一种细胞高含量免疫荧光HBV核心检测方法，以鉴定诱导

StephenW

Development of a cellular high-content, immunofluorescent HBV core assay to identify novel capsid assembly modulators that induce the formation of aberrant HBV core structures
Sarah Sauviller  1 , Karen Vergauwen  1 , Steffen Jaensch  1 , Emmanuel Gustin  1 , Danielle Peeters  1 , Peter Vermeulen  1 , Dirk Wuyts  1 , Koen Vandyck  1 , Frederik Pauwels  1 , Jan Martin Berke  2
Affiliations
Affiliations

    1
    Janssen Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium.
    2
    Janssen Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium. Electronic address: [email protected].

    PMID: 33839187 DOI: 10.1016/j.jviromet.2021.114150

Abstract

Hepatitis B Virus (HBV) core protein has multiple functions in the viral life cycle and is an attractive target for new anti-viral therapies. Capsid assembly modulators (CAMs) target the core protein and induce the formation of either morphologically normal (CAM-N) or aberrant structures (CAM-A), both devoid of genomic material. To date a diverse family of CAM-N chemotypes has been identified, but in contrast, described CAM-As are based on the heteroaryldihydropyrimidine (HAP) scaffold. We used the HBV-inducible HepG2.117 cell line with immunofluorescent labeling of HBV core to develop and validate a cellular high-content image-based assay where aggregated core structures are identified using image analysis spot texture features. Treatment with HAPs led to a dose- and time-dependent formation of aggregated core appearing as dot-like structures in the cytoplasm and nucleus. By combining a biochemical and cellular screening approach, a compound was identified as a novel non-HAP scaffold able to induce dose-dependent formation of aberrant core structures, which was confirmed by electron microscopy and native gel electrophoresis. This compound displayed anti-HBV activity in HepG2.117 cells, providing proof-of-concept for our screening approach. We believe our combined biochemical and cellular high-content screening method will aid in expanding the range of CAM-A chemotypes.

Keywords: HBV core; aberrant core structures; capsid assembly modulator; hepatitis B; high-content; non-HAP scaffold.

Copyright © 2021. Published by Elsevier B.V.

StephenW

开发一种细胞高含量免疫荧光HBV核心检测方法，以鉴定诱导异常HBV核心结构形成的新型衣壳装配调节剂
莎拉·索维尔（Sarah Sauviller）1，卡伦·韦高（Karen Vergauwen）1，斯特芬·詹斯（Steffen Jaensch）1，伊曼纽尔·古斯汀（Emmanuel Gustin）1，丹妮尔·皮特斯（Danielle Peeters）1，彼得·维米尔（Peter Vermeulen）1，德克·伍兹（Dirk Wuyts）1，科恩·范迪克（Koen Vandyck）1，弗雷德里克·鲍威尔（Frederik Pauwels）1，扬·马丁·伯克（Jan Martin Berke）2
隶属关系
隶属关系

    1个
    Janssen Research and Development，Turnhoutseweg 30，比利时比尔塞2340。
    2个
    Janssen Research and Development，Turnhoutseweg 30，比利时比尔塞2340。电子地址：[email protected]。

    PMID：33839187 DOI：10.1016 / j.jviromet.2021.114150

抽象的

乙型肝炎病毒（HBV）核心蛋白在病毒生命周期中具有多种功能，是新的抗病毒治疗的有吸引力的靶标。衣壳装配调节剂（CAM）靶向核心蛋白并诱导形态正常的（CAM-N）或异常结构（CAM-A）的形成，两者均不含基因组物质。迄今为止，已经鉴定出多种多样的CAM-N化学型家族，但是相反，所描述的CAM-A基于杂芳基二氢嘧啶（HAP）支架。我们使用具有HBV核心免疫荧光标记功能的HBV诱导性HepG2.117细胞系来开发和验证基于细胞高含量图像的检测方法，其中使用图像分析点纹理特征来识别聚集的核心结构。用HAPs治疗导致聚集核的剂量和时间依赖性形成，其在细胞质和细胞核中以点状结构出现。通过结合生物化学和细胞筛选方法，该化合物被鉴定为能够诱导剂量依赖性异常核心结构形成的新型非HAP支架，这已通过电子显微镜和天然凝胶电泳得以证实。该化合物在HepG2.117细胞中显示抗HBV活性，为我们的筛选方法提供了概念验证。我们相信，将生化和细胞高含量筛选相结合的方法将有助于扩大CAM-A化学型的范围。

关键字：HBV核心；异常的核心结构；衣壳装配调节剂；乙型肝炎；高含量非HAP支架。

版权所有©2021，由Elsevier B.V.发布。