PDF(Pubmed)
Abstract：
UNASSIGNED: Chronic hepatitis delta is the most severe form of chronic viral hepatitis and is associated with faster progression towards cirrhosis, liver decompensation, and hepatocellular carcinoma. Hepatitis delta virus (HDV)\'s tight dependency on hepatitis B virus and the host cell machinery for its life cycle limits the development of direct-acting antivirals. Thus, we aimed to identify compounds that could block HDV replication by targeting its antigenomic ribozyme.
UNASSIGNED: We generated stable Huh7 human hepatoma cells expressing a reporter gene (Gaussia luciferase) either downstream (Gluc-2xRz) or upstream (2xRz-Gluc) of two HDV antigenomic ribozyme sequences. We performed high-throughput screening of three small molecule libraries. The secreted luciferase was measured as a readout of ribozyme inhibition upon addition of the molecules. Each plate was considered valid when the Z factor was >0.4. Specificity and toxicity evaluations were performed for the hits with a Z-score >5 and half-maximal inhibitory concentration was calculated by performing a dose-response experiment.
UNASSIGNED: A dose-dependent induction of luciferase expression was detected in Gluc-2xRz-transfected cells incubated with the antisense morpholino, suggesting that the catalytic activity of the ribozyme cloned downstream of the reporter gene was efficiently inhibited. Among the 6,644 compounds screened, we identified four compounds that showed a specific inhibitory effect on the HDV antigenomic ribozyme in Gluc-2xRz cells, i.e. three histone deacetylase inhibitors and the purine analogue 8-azaguanine. The latter also significantly decreased HDV replication (by 40%) in differentiated HepaRG cells six days post infection.
UNASSIGNED: Using a novel cell culture model, we identified four small molecules active against the antigenomic HDV ribozyme. These results may provide insights into the structural requirements of molecules designed for the potent and specific inhibition of HDV replication.
UNASSIGNED: Chronic hepatitis delta is the most severe form of chronic viral hepatitis and is associated with faster progression towards cirrhosis, liver decompensation, and the development of hepatocellular carcinoma. Despite the current development of several new compounds, there is still a need for efficient antiviral treatments specifically targeting hepatitis delta virus (HDV). This work describes a novel cell culture model that allows for the high-throughput screening of compounds able to inhibit HDV ribozymes. We identified four small molecules active against the antigenomic HDV ribozyme (the ribozyme involved in the early step of HDV replication), with the strongest activity shown by 8-azaguanine, a purine analogue. Our data may provide insights into the structural requirements of molecules designed to inhibit HDV.
UNASSIGNED: We generated stable Huh7 human hepatoma cells expressing a reporter gene (Gaussia luciferase) either downstream (Gluc-2xRz) or upstream (2xRz-Gluc) of two HDV antigenomic ribozyme sequences. We performed high-throughput screening of three small molecule libraries. The secreted luciferase was measured as a readout of ribozyme inhibition upon addition of the molecules. Each plate was considered valid when the Z factor was >0.4. Specificity and toxicity evaluations were performed for the hits with a Z-score >5 and half-maximal inhibitory concentration was calculated by performing a dose-response experiment.
UNASSIGNED: A dose-dependent induction of luciferase expression was detected in Gluc-2xRz-transfected cells incubated with the antisense morpholino, suggesting that the catalytic activity of the ribozyme cloned downstream of the reporter gene was efficiently inhibited. Among the 6,644 compounds screened, we identified four compounds that showed a specific inhibitory effect on the HDV antigenomic ribozyme in Gluc-2xRz cells, i.e. three histone deacetylase inhibitors and the purine analogue 8-azaguanine. The latter also significantly decreased HDV replication (by 40%) in differentiated HepaRG cells six days post infection.
UNASSIGNED: Using a novel cell culture model, we identified four small molecules active against the antigenomic HDV ribozyme. These results may provide insights into the structural requirements of molecules designed for the potent and specific inhibition of HDV replication.
UNASSIGNED: Chronic hepatitis delta is the most severe form of chronic viral hepatitis and is associated with faster progression towards cirrhosis, liver decompensation, and the development of hepatocellular carcinoma. Despite the current development of several new compounds, there is still a need for efficient antiviral treatments specifically targeting hepatitis delta virus (HDV). This work describes a novel cell culture model that allows for the high-throughput screening of compounds able to inhibit HDV ribozymes. We identified four small molecules active against the antigenomic HDV ribozyme (the ribozyme involved in the early step of HDV replication), with the strongest activity shown by 8-azaguanine, a purine analogue. Our data may provide insights into the structural requirements of molecules designed to inhibit HDV.
摘要：
未经证实：慢性丁型肝炎是慢性病毒性肝炎的最严重形式，与肝硬化的更快进展有关。肝脏代偿失调,和肝细胞癌。丁型肝炎病毒（HDV）对乙型肝炎病毒和其生命周期的宿主细胞机制的紧密依赖性限制了直接作用抗病毒药物的发展。因此，我们的目的是鉴定可以通过靶向其反基因组核酶来阻断HDV复制的化合物。
UNASSIGNED:我们产生了稳定的Huh7人肝癌细胞,其表达两个HDV反基因组核酶序列的下游(Gluc-2xRz)或上游(2xRz-Gluc)的报告基因(高斯荧光素酶)。我们对三个小分子文库进行了高通量筛选。将分泌的荧光素酶测量为添加分子后的核酶抑制的读数。当Z因子>0.4时,每个板被认为是有效的。对Z评分>5的命中进行特异性和毒性评价,并通过进行剂量反应实验计算半数最大抑制浓度。
UNASSIGNED：在与反义吗啉代孵育的Gluc-2xRz转染细胞中检测到剂量依赖性的荧光素酶表达诱导，这表明报道基因下游克隆的核酶的催化活性被有效抑制。在筛选的6644种化合物中，我们鉴定了四种化合物，它们对Gluc-2xRz细胞中的HDV反基因组核酶具有特定的抑制作用，即三种组蛋白脱乙酰酶抑制剂和嘌呤类似物8-氮杂鸟嘌呤。后者还在感染后6天显著降低了分化的HepaRG细胞中的HDV复制(40%)。
未经评估：使用一种新的细胞培养模型，我们鉴定了4个对反基因组HDV核酶有活性的小分子.这些结果可以提供对设计用于有效和特异性抑制HDV复制的分子的结构要求的见解。
UNASSIGNED：慢性丁型肝炎是慢性病毒性肝炎的最严重形式，与更快的肝硬化进展有关。肝脏代偿失调,和肝细胞癌的发展。尽管目前开发了几种新化合物，仍然需要专门针对丁型肝炎病毒(HDV)的有效抗病毒治疗.这项工作描述了一种新颖的细胞培养模型，该模型允许高通量筛选能够抑制HDV核酶的化合物。我们确定了四种对反基因组HDV核酶（参与HDV复制早期步骤的核酶）有活性的小分子，8-azaguanine显示出最强的活性，嘌呤类似物.我们的数据可以提供对旨在抑制HDV的分子的结构要求的见解。
UNASSIGNED:我们产生了稳定的Huh7人肝癌细胞,其表达两个HDV反基因组核酶序列的下游(Gluc-2xRz)或上游(2xRz-Gluc)的报告基因(高斯荧光素酶)。我们对三个小分子文库进行了高通量筛选。将分泌的荧光素酶测量为添加分子后的核酶抑制的读数。当Z因子>0.4时,每个板被认为是有效的。对Z评分>5的命中进行特异性和毒性评价,并通过进行剂量反应实验计算半数最大抑制浓度。
UNASSIGNED：在与反义吗啉代孵育的Gluc-2xRz转染细胞中检测到剂量依赖性的荧光素酶表达诱导，这表明报道基因下游克隆的核酶的催化活性被有效抑制。在筛选的6644种化合物中，我们鉴定了四种化合物，它们对Gluc-2xRz细胞中的HDV反基因组核酶具有特定的抑制作用，即三种组蛋白脱乙酰酶抑制剂和嘌呤类似物8-氮杂鸟嘌呤。后者还在感染后6天显著降低了分化的HepaRG细胞中的HDV复制(40%)。
未经评估：使用一种新的细胞培养模型，我们鉴定了4个对反基因组HDV核酶有活性的小分子.这些结果可以提供对设计用于有效和特异性抑制HDV复制的分子的结构要求的见解。
UNASSIGNED：慢性丁型肝炎是慢性病毒性肝炎的最严重形式，与更快的肝硬化进展有关。肝脏代偿失调,和肝细胞癌的发展。尽管目前开发了几种新化合物，仍然需要专门针对丁型肝炎病毒(HDV)的有效抗病毒治疗.这项工作描述了一种新颖的细胞培养模型，该模型允许高通量筛选能够抑制HDV核酶的化合物。我们确定了四种对反基因组HDV核酶（参与HDV复制早期步骤的核酶）有活性的小分子，8-azaguanine显示出最强的活性，嘌呤类似物.我们的数据可以提供对旨在抑制HDV的分子的结构要求的见解。
