Discovery of New Hepatitis B Virus Capsid Assembly Modulators by an Optimal High-Throughput Cell-Based Assay
Discovery of New Hepatitis B Virus Capsid Assembly Modulators by an Optimal High-Throughput Cell-Based Assay
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通过最佳高通量细胞检测发现新型乙型肝炎病毒衣壳组装调节剂
DOI：
10.1021/acsinfecdis.9b00030
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发表时间：
2019-05-01
影响因子：
5.3
通讯作者：
Liu, Gang
中科院分区：
文献类型：
--
作者：
Pei, Yameng;Wang, Chunting;Liu, Gang
本文建立了一种简单有效的高通量筛选(HTS)方法，利用HepAD38荧光素酶报告基因(HepAD38-Luc)细胞株，有效地排除了针对四环素关闭(TETOFF)调控系统的假阳性化合物。通过筛选内部化学文库，鉴定出1和2代表的N-苯基哌啶-3-甲酰胺衍生物，同时排除了其他假阳性化合物(即喹恶啉(3)和苯并噻嗪(4)衍生物)。化合物1和2在HepAD38和HepG2.2.15细胞中均表现出较强的抑制乙肝病毒复制的活性。进一步的研究表明，1和2降低了细胞外的HBVDNA、HBeAg和细胞内的HBVDNA中间体，包括总DNA、RNA和前C区RNA。大小排斥色谱(SEC)和电子显微镜(EM)研究表明，1和2显著诱导了形态完整的衣壳的形成，并加速了衣壳组装的动力学，表明1和2都是I型衣壳组装调节剂(CAM)。
In this article, a simple and effective high-throughput screening (HTS) assay was developed to identify anti-HBV compounds by using a HepAD38 luciferase reporter (HepAD38-luc) cell line that can effectively exclude the false positive hit compounds targeted on the tetracycline off (tetoff) regulation system. Through screening in-house chemical libraries, N-phenylpiperidine-3-carboxamide derivatives, represented by 1 and 2, were identified, while the other false positive hits (i.e., quinoxaline (3) and benzothiazin (4) derivatives) were simultaneously excluded. Compounds 1 and 2 exhibit strong inhibitory activity against HBV replication in both HepAD38 and HepG2.2.15 cells. Further studies revealed that 1 and 2 reduced extracellular HBV DNA, HBeAg, and intracellular HBV intermediates, including total DNA, RNA, and precore RNA of HBV. Size-exclusion chromatography (SEC) and electron microscopy (EM) investigations demonstrated that 1 and 2 remarkably induced the formation of morphologically intact capsids and accelerated the dynamics of capsid assembly, suggesting that both 1 and 2 were type I capsid assembly modulators (CAMs).