Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay
Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay
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通过体外聚合酶延伸测定鉴定非核苷乙型肝炎病毒聚合酶抑制剂
DOI：
10.1007/s00535-019-01643-0
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发表时间：
2019
影响因子：
6.3
通讯作者：
Toyoda Tetsuya
中科院分区：
文献类型：
--
作者：
Nakajima Shogo;Watashi Koichi;Fukano Kento;Tsukuda Senko;Wakae Kousho;Aizaki Hideki;Muramatsu Masamichi;Wakita Takaji;Toyoda Tetsuya
研究背景B型肝炎病毒（HBV）聚合酶是HBV基因组形成过程中唯一必需的酶，是一个很有吸引力的药物靶点。然而，合成和纯化重组HBV聚合酶蛋白的困难阻碍了靶向该酶的新药的开发，特别是与核苷结构无关的化合物。我们最近开发了一种合成和纯化重组HBV聚合酶的技术，该聚合酶含有逆转录酶（RT）结构域，具有体外DNA延伸活性。方法我们使用过量产生的蛋白质建立了体外高通量筛选系统，以鉴定抑制HBV聚合酶延伸活性的化合物。作为一种潜在的抗HBV药物。衍生物分析鉴定了另一种芪衍生物PDM 2，其能够抑制HBV复制，IC 50为14.4 ± 7.7 μM。一项感染实验表明，这些化合物抑制HBV的复制，而不是像预期的那样抑制进入过程。表面等离子体共振分析表明PDM 2和RT结构域之间的特异性相互作用。重要的是，PDM 2对野生型HBV和拉米夫定/恩替卡韦耐药HBV变异体的复制显示出相似的抑制活性。此外，PDM 2显示出与临床上使用的nucleos（t）ide analogs.ConclusionsWe报告的筛选系统，这是用于识别非nucleos（t）ide RT抑制剂的发展的累加效应。
BackgroundHepatitis B virus (HBV) polymerase is the only virus-encoded enzyme essential for producing the HBV genome and is regarded as an attractive drug target. However, the difficulty of synthesizing and purifying recombinant HBV polymerase protein has hampered the development of new drugs targeting this enzyme, especially compounds unrelated to the nucleoside structure. We recently have developed a technique for the synthesis and purification of recombinant HBV polymerase containing the reverse transcriptase (RT) domain that carried DNA elongation activity in vitro.MethodsWe used the overproduced protein to establish an in vitro high-throughput screening system to identify compounds that inhibit the elongation activity of HBV polymerase.ResultsWe screened 1120 compounds and identified a stilbene derivative, piceatannol, as a potential anti-HBV agent. Derivative analysis identified another stilbene derivative, PDM2, that was able to inhibit HBV replication with an IC50of 14.4 ± 7.7 μM. An infection experiment suggested that the compounds inhibit the replication of HBV rather than the entry process, as expected. Surface plasmon resonance analysis demonstrated a specific interaction between PDM2 and the RT domain. Importantly, PDM2 showed similar inhibitory activity against the replication of both wild-type HBV and a lamivudine/entecavir-resistant HBV variant. Furthermore, PDM2 showed an additive effect in combination with clinically used nucleos(t)ide analogs.ConclusionsWe report the development of a screening system that is useful for identifying non-nucleos(t)ide RT inhibitors.