Establishment of a system for finding inhibitors of ε RNA binding with the HBV polymerase
Establishment of a system for finding inhibitors of ε RNA binding with the HBV polymerase
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建立寻找与 HBV 聚合酶结合的 ε RNA 抑制剂的系统
DOI：
10.1111/gtc.12778
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发表时间：
2020
期刊：
影响因子：
2.1
通讯作者：
Ueda Keiji
中科院分区：
文献类型：
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作者：
Liu Xiao‐Quan;Ohsaki Eriko;Ueda Keiji
虽然有几种核苷类似物可用于治疗HBV感染，但长期使用这些药物治疗可能导致耐药病毒的出现。最近的HIV-1研究表明，使用核苷酸逆转录酶抑制剂（NRTI）和非核苷酸逆转录酶抑制剂（NNRTI）的联合治疗可以显著抑制NRTI耐药病毒的病毒基因组复制。为实现HBV的联合治疗，需要开发新的NRTI和NNRTI。在这里，我们的目的是确定靶向HBV聚合酶末端蛋白（TP）-逆转录酶（RT）（TP-RT）结构域的新型NNRTI，这是HBV复制的关键结构域。我们利用大肠杆菌表达系统表达并纯化了高纯度的HBV TP‐RT，并建立了体外ε RNA结合试验系统。然后，我们在无细胞试验中使用TP‐RT从化合物库中筛选候选抑制剂，并鉴定了两种化合物，6-羟基-DL‐DOPA和N‐油酰多巴胺，它们抑制ε RNA与HBV聚合酶的结合。此外，这些药物还通过抑制前基因组RNA包装到衣壳中来降低基于细胞的测定中的HBV DNA水平。本文开发的新型筛选系统应该为发现靶向HBV TP‐RT结构域的药物以治疗HBV感染开辟一条新途径。
Although several nucleo(s)tide analogs are available for treatment of HBV infection, long‐term treatment with these drugs can lead to the emergence of drug‐resistant viruses. Recent HIV‐1 studies suggest that combination therapies using nucleo(s)tide reverse transcriptase inhibitors (NRTIs) and non‐nucleo(s)tide reverse transcriptase inhibitors (NNRTIs) could drastically inhibit the viral genome replication of NRTI‐resistant viruses. In order to carry out such combinational therapy against HBV, several new NRTIs and NNRTIs should be developed. Here, we aimed to identify novel NNRTIs targeting the HBV polymerase terminal protein (TP)‐reverse transcriptase (RT) (TP‐RT) domain, which is a critical domain for HBV replication. We expressed and purified the HBV TP‐RT with high purity using anEscherichia coliexpression system and established an in vitro ε RNA‐binding assay system. Then, we used TP‐RT in cell‐free assays to screen candidate inhibitors from a chemical compound library, and identified two compounds, 6‐hydroxy‐DL‐DOPA and N‐oleoyldopamine, which inhibited the binding of ε RNA with the HBV polymerase. Furthermore, these drugs reduced HBV DNA levels in cell‐based assays as well by inhibiting packaging of pregenome RNA into capsids. The novel screening system developed herein should open a new pathway the discovery of drugs targeting the HBV TP‐RT domain to treat HBV infection.