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Identification and characterization of a novel hepatitis B virus pregenomic RNA encapsidation inhibitor.
Antiviral Research ( IF 7.6 ) Pub Date : 2020-01-12 , DOI: 10.1016/j.antiviral.2020.104709 Eunji Jo 1 , Dong-Kyun Ryu 1 , Alexander König 1 , Soonju Park 2 , Yoojin Cho 1 , Sang-Hyun Park 1 , Tae-Hee Kim 2 , Seung Kew Yoon 3 , Wang-Shick Ryu 4 , Jonathan Cechetto 2 , Marc P Windisch 5
Antiviral Research ( IF 7.6 ) Pub Date : 2020-01-12 , DOI: 10.1016/j.antiviral.2020.104709 Eunji Jo 1 , Dong-Kyun Ryu 1 , Alexander König 1 , Soonju Park 2 , Yoojin Cho 1 , Sang-Hyun Park 1 , Tae-Hee Kim 2 , Seung Kew Yoon 3 , Wang-Shick Ryu 4 , Jonathan Cechetto 2 , Marc P Windisch 5
Affiliation
Currently, therapies to treat chronic hepatitis B (CHB) infection are based on the use of interferon-α or nucleos(t)ide analogs (NAs) to prevent viral DNA synthesis by inhibiting the reverse transcriptase activity of the hepatitis B virus (HBV) polymerase (Pol). However, these therapies are not curative; thus, the development of novel anti-HBV agents is needed. In accordance with this unmet medical need, we devised a new target- and cell-based, high-throughput screening assay to identify novel small molecules that block the initial interaction of the HBV Pol with its replication template the viral pregenomic RNA (pgRNA). We screened approximately 110,000 small molecules for the ability to prevent HBV Pol recognition of the pgRNA 5' epsilon (ε) stem-loop structure, identifying (Z)-2-(allylamino)-4-amino-N'-cyanothiazole-5-carboximidamide (AACC). Viral nucleocapsid-captured quantitative RT-PCR and Western blot results revealed that AACC significantly decreased encapsidated pgRNA levels and blocked capsid assembly without affecting core protein expression in stable HBV-replicating cells. As a result, both intra- and extracellular accumulation of viral DNA was strongly reduced. AACC treatment of HepG2-sodium taurocholate transporting polypeptide (NTCP) cells and primary human hepatocytes infected with cell culture- or patient-derived HBV isolates showed both time- and dose-dependent inhibition of infectious viral progeny and rcDNA production. Furthermore, AACC showed cross-genotypic activity against genotypes B, C, and D. Of note, AACC inhibited the viral replication of lamivudine and a capsid inhibitor-resistant HBV, and showed synergistic effects with NAs and a capsid inhibitor. In conclusion, we identified a novel class of compounds specifically targeting the ε-Pol interaction and thereby preventing the encapsidation of pgRNAs into viral capsids. This promising new HBV inhibitor class potently inhibits HBV amplification with distinct characteristics from existing NAs and other drugs currently under development, promising to add value to existing therapies for CHB.
中文翻译:
新型乙肝病毒前基因组RNA衣壳化抑制剂的鉴定和表征。
当前,用于治疗慢性乙型肝炎(CHB)感染的疗法是基于干扰素-α或核苷酸(t)核苷酸类似物(NAs)的使用,通过抑制乙型肝炎病毒(HBV)的逆转录酶活性来防止病毒DNA合成聚合酶(Pol)。然而,这些疗法不是治愈的。因此,需要开发新型抗HBV药物。根据未满足的医疗需求,我们设计了一种新的基于靶标和细胞的高通量筛选测定方法,以鉴定能够阻断HBV Pol及其复制模板与病毒前基因组RNA(pgRNA)初始相互作用的新型小分子。我们筛选了大约110,000个小分子以防止HBV Pol识别pgRNA 5'ε(ε)茎环结构,从而识别(Z)-2-(烯丙胺基)-4-氨基-N' -氰基噻唑-5-羧酰亚胺(AACC)。病毒核衣壳捕获的定量RT-PCR和Western印迹结果表明,AACC显着降低了衣壳pgRNA的水平并阻止了衣壳装配,而没有影响稳定的HBV复制细胞中的核心蛋白表达。结果,病毒DNA的细胞内和细胞外积累都大大减少了。AACC处理的牛肝胆碱转运蛋白多肽(NTCP)细胞和原代人肝细胞感染了细胞培养物或患者来源的HBV分离株,对感染性病毒后代和rcDNA的产生均表现出时间和剂量依赖性。此外,AACC显示出对基因型B,C和D的交叉基因型活性。值得注意的是,AACC抑制了拉米夫定和衣壳抑制剂耐药性HBV的病毒复制,并显示出与NAs和衣壳抑制剂的协同作用。总而言之,我们鉴定了一类新的化合物,这些化合物专门针对ε-Pol相互作用,从而防止pgRNA衣壳化为病毒衣壳。这种有前途的新型HBV抑制剂可有效抑制HBV扩增,具有与现有NAs和目前正在开发的其他药物不同的特征,有望为CHB的现有疗法增加价值。
更新日期:2020-01-13
中文翻译:
新型乙肝病毒前基因组RNA衣壳化抑制剂的鉴定和表征。
当前,用于治疗慢性乙型肝炎(CHB)感染的疗法是基于干扰素-α或核苷酸(t)核苷酸类似物(NAs)的使用,通过抑制乙型肝炎病毒(HBV)的逆转录酶活性来防止病毒DNA合成聚合酶(Pol)。然而,这些疗法不是治愈的。因此,需要开发新型抗HBV药物。根据未满足的医疗需求,我们设计了一种新的基于靶标和细胞的高通量筛选测定方法,以鉴定能够阻断HBV Pol及其复制模板与病毒前基因组RNA(pgRNA)初始相互作用的新型小分子。我们筛选了大约110,000个小分子以防止HBV Pol识别pgRNA 5'ε(ε)茎环结构,从而识别(Z)-2-(烯丙胺基)-4-氨基-N' -氰基噻唑-5-羧酰亚胺(AACC)。病毒核衣壳捕获的定量RT-PCR和Western印迹结果表明,AACC显着降低了衣壳pgRNA的水平并阻止了衣壳装配,而没有影响稳定的HBV复制细胞中的核心蛋白表达。结果,病毒DNA的细胞内和细胞外积累都大大减少了。AACC处理的牛肝胆碱转运蛋白多肽(NTCP)细胞和原代人肝细胞感染了细胞培养物或患者来源的HBV分离株,对感染性病毒后代和rcDNA的产生均表现出时间和剂量依赖性。此外,AACC显示出对基因型B,C和D的交叉基因型活性。值得注意的是,AACC抑制了拉米夫定和衣壳抑制剂耐药性HBV的病毒复制,并显示出与NAs和衣壳抑制剂的协同作用。总而言之,我们鉴定了一类新的化合物,这些化合物专门针对ε-Pol相互作用,从而防止pgRNA衣壳化为病毒衣壳。这种有前途的新型HBV抑制剂可有效抑制HBV扩增,具有与现有NAs和目前正在开发的其他药物不同的特征,有望为CHB的现有疗法增加价值。