■慢性乙型肝炎是一种不治之症。由于缺乏合适的细胞培养模型来研究单一实验装置中的HBV生命周期,因此无法解决未满足的医疗治疗需求。我们试图开发一个适合调查整个HBV生命周期的各个方面的平台。
■用HBV接种HepG2-NTCPsec+细胞。将受感染细胞的上清液转移至原初细胞。通过病毒和细胞因子的高含量成像确定在原代和继发性感染细胞中的感染抑制。新型抗病毒药物在感染细胞培养或患者来源的HBV的细胞和稳定的病毒复制细胞中进行分类。进行HBV内化和基于靶标的受体结合测定。
■我们开发了一个HBV平台,筛选了2,102种药物和生物活性物质,并使用感染性HBV基因型D确定了3种早期和38种晚期新型HBV生命周期抑制剂,普朗司特(EC504.3μM;50%细胞毒性浓度[CC50]>50μM)和细胞松弛素D(EC500.07μM;CC50>50μM),和2种晚期抑制剂,氟达拉滨(EC500.1μM;CC5013.4μM)和右美托咪定(EC506.2μM;CC50>50μM),进一步调查。普卢司特抑制HBVpreS1结合,而细胞松弛素D阻止HBV的内在化。氟达拉滨抑制HBV后代DNA的分泌,而右美托咪定干扰HBV后代的感染性。患者来源的HBV基因型C被氟达拉滨(EC500.08μM)和右美托咪定(EC508.7μM)有效抑制。
■新开发的高含量测定适用于筛选大型药物库,能够监测整个HBV生命周期,并区分病毒生命周期早期和晚期事件的抑制。
■HBV感染是无法治愈的,几乎没有可用的治疗方法的慢性疾病。由于缺乏合适的细胞培养模型以在单个实验设置中研究整个病毒生命周期,因此无法满足这种未满足的医疗需求。我们开发了一种基于图像的方法,适用于筛选大量药物,使用可以被HBV感染并产生大量病毒颗粒的细胞系。通过将病毒上清液从这些受感染的细胞转移到未感染的靶细胞,我们可以监测整个病毒生命周期。我们使用这个系统来筛选药物库,并确定了新的抗HBV抑制剂,在其生命周期的各个阶段有效抑制HBV。该测定将是研究HBV生命周期和加速新型治疗策略开发的重要新工具。
UNASSIGNED: Chronic hepatitis B is an incurable disease. Addressing the unmet medical need for therapies has been hampered by a lack of suitable cell culture models to investigate the HBV life cycle in a single experimental setup. We sought to develop a platform suitable to investigate all aspects of the entire HBV life cycle.
UNASSIGNED: HepG2-NTCPsec+ cells were inoculated with HBV. Supernatants of infected cells were transferred to naïve cells. Inhibition of infection was determined in primary and secondary infected cells by high-content imaging of viral and cellular factors. Novel antivirals were triaged in cells infected with cell culture- or patient-derived HBV and in stably virus replicating cells. HBV internalisation and target-based receptor binding assays were conducted.
UNASSIGNED: We developed an HBV platform, screened 2,102 drugs and bioactives, and identified 3 early and 38 late novel HBV life cycle inhibitors using infectious HBV genotype D. Two early inhibitors, pranlukast (EC50 4.3 μM; 50% cytotoxic concentration [CC50] >50 μM) and cytochalasin D (EC50 0.07 μM; CC50 >50 μM), and 2 late inhibitors, fludarabine (EC50 0.1 μM; CC50 13.4 μM) and dexmedetomidine (EC50 6.2 μM; CC50 >50 μM), were further investigated. Pranlukast inhibited HBV preS1 binding, whereas cytochalasin D prevented the internalisation of HBV. Fludarabine inhibited the secretion of HBV progeny DNA, whereas dexmedetomidine interfered with the infectivity of HBV progeny. Patient-derived HBV genotype C was efficiently inhibited by fludarabine (EC50 0.08 μM) and dexmedetomidine (EC50 8.7 μM).
UNASSIGNED: The newly developed high-content assay is suitable to screen large-scale drug libraries, enables monitoring of the entire HBV life cycle, and discriminates between inhibition of early and late viral life cycle events.
UNASSIGNED: HBV infection is an incurable, chronic disease with few available treatments. Addressing this unmet medical need has been hampered by a lack of suitable cell culture models to study the entire viral life cycle in a single experimental setup. We developed an image-based approach suitable to screen large numbers of drugs, using a cell line that can be infected by HBV and produces large amounts of virus particles. By transferring viral supernatants from these infected cells to uninfected target cells, we could monitor the entire viral life cycle. We used this system to screen drug libraries and identified novel anti-HBV inhibitors that potently inhibit HBV in various phases of its life cycle. This assay will be an important new tool to study the HBV life cycle and accelerate the development of novel therapeutic strategies.