乙肝病毒核糖核酸酶H作为药物靶标

StephenW

Review
The hepatitis B virus ribonuclease H as a drug target

    John E. Tavis, , Elena Lomonosova

    Department of Molecular Microbiology and Immunology, Saint Louis University Liver Center, United States

    Received 18 February 2015, Revised 30 March 2015, Accepted 2 April 2015, Available online 8 April 2015

Highlights

•    The HBV ribonuclease H is a promising but unexploited drug target.
•    Recent technical advances permit low-throughput screening for ribonuclease H inhibitors.
•    Some of the known HIV ribonuclease H inhibitors also inhibit HBV.
•    Combining ribonuclease H inhibitors with existing or novel drugs may significantly improve treatment efficacy for HBV.

Abstract

Chronic hepatitis B virus (HBV) infection is a leading cause of hepatitis, liver failure, and hepatocellular carcinoma. An outstanding vaccine is available; however, the number of infections remains high. Current anti-HBV treatments with interferon α and nucleos(t)ide analogs clear the infection in only a small minority of patients, and either induce serious side-effects or are of very long duration. HBV is a small, enveloped DNA virus that replicates by reverse transcription via an RNA intermediate. The HBV ribonuclease H (RNaseH) is essential for viral replication, but it has not been exploited as a drug target. Recent low-throughput screening of compound classes with anti-Human Immunodeficiency Virus RNaseH activity led to identification of HBV RNaseH inhibitors in three different chemical families that block HBV replication. These inhibitors are promising candidates for development into new anti-HBV drugs. The RNaseH inhibitors may help improve treatment efficacy enough to clear the virus from the liver when used in combination with existing anti-HBV drugs and/or with other novel inhibitors under development. This article forms part of a symposium in Antiviral Research on “An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B.”
Keywords

    Hepatitis B virus; Ribonuclease H; Reverse transcription; Inhibitors

StephenW

回顾
乙肝病毒核糖核酸酶H作为药物靶标

    约翰·塔维斯，埃琳娜Lomonosova

    分子微生物学和免疫学，圣路易斯大学肝脏中心，美国科

    收到2015年2月18日，修订二〇一五年三月三十零日，接受2015年4月2日，可在线2015年4月8日

亮点

•在乙肝病毒核糖核酸酶H是一种很有前途，但尚未开发的药物靶标。
•最近的技术进步允许低通量筛选核糖核酸酶H抑制剂。
•一些已知的艾滋病病毒核糖核酸酶H抑制剂也抑制HBV。
•组合核糖核酸酶H抑制剂与现有或新的药物可能显著提高治疗疗效乙肝。

抽象

慢性乙型肝炎病毒（HBV）感染是肝炎，肝功能衰竭和肝细胞癌的主要病因。一个突出的疫苗可用;然而，感染人数仍然很高。当前的抗乙肝病毒治疗用干扰素α和核苷（酸）类似物清除感染的只有一小部分患者，无论是和发生严重的副作用或者是持续时间很长的。乙型肝炎是一种小型，包膜DNA病毒，它通过一种RNA中间复制逆转录。乙肝病毒核糖核酸酶H（RNA酶H）是病毒复制所必需的，但它并没有被利用作为药物靶标。化合物类的抗人免疫缺陷病毒RNA酶H活性的近期低通量筛选导致鉴定HBV的RNA酶H抑制剂在三个不同的化学族，阻止HBV复制。这些抑制剂是有希望的候选者发展成新的抗HBV药物。的RNA酶H抑制剂可有助于提高治疗功效足够清除病毒从肝脏中组合使用与现有的抗HBV药物和/或与其它正在开发的新型抑制剂时。本文形成在抗病毒研究的专题讨论会的一部分，“一个未完成的故事：从澳大利亚抗原的发现到新的治疗疗法的发展为乙型肝炎”
关键词

    乙型肝炎病毒;核糖核酸酶H;反转录;抑制剂

StephenW

6. Known anti-HBV RNaseH compounds

Over 190 compounds in the αHT, N-hydroxyisoquinolinedione, napthyridinone, dioxobutanoic acid, hydroxychromenone, Elvitegravir, Raltegravir, aminocyanothiophene, hydroxyxanthenone, cyanopyran, and thienopyrimidinone chemical families have been screened against the HBV RNaseH. Nineteen compounds inhibited the HBV RNaseH at ⩽20 μM, with the best hits having low micromolar IC50 values [(Cai et al., 2014, Hu et al., 2013, Lu et al., 2015 and Tavis et al., 2013) and unpublished]. Strong hits were observed among the αHTs, N-hydroxyisoquinolinediones, napthyridinones, and hydroxychomenones. In all cases, the compounds had similar activities against HBV genotypes C, D, and/or H. The inhibitors appear to act by chelating the divalent cations in the RNaseH active site because removing one of the three Lewis basic moieties or altering their angles relative to each other ablates inhibition [(Cai et al., 2014 and Lu et al., 2015) and unpublished]. Counter-screening against human RNaseH1 revealed that most of these screening hits were also active against the human enzyme, but differences in the sensitivity of the two enzymes were apparent (Lu et al., 2015). This indicates that increasing selectivity for the HBV enzyme must be a major focus during chemical optimization.

Thirty-seven compounds have been tested for ability to suppress HBV replication in culture; 10 inhibited HBV replication at ⩽20 μM (Table 1). Preferential suppression of the viral plus-polarity DNA strand confirmed that inhibition was due to blocking the viral RNaseH activity for all compounds except #56. Moderate cytotoxicity was observed for all active compounds, with CC50 values ranging from 6.1 to 79 μM. Therefore, it will be essential to minimize cytotoxicity during development of RNaseH inhibitors. The EC50 values for most of these inhibitors was substantially lower than their IC50 values. The cause for this quantitative discrepancy is unknown, but it could be due to accumulation of the compounds in cells, metabolism of the inhibitors to more active forms, or most likely, that the recombinant RNaseH does not recapitulate all aspects of the enzyme in its natural context as part of the full-length HBV polymerase protein. Nevertheless, strong inhibition of the recombinant enzyme is an excellent predictor of a compound’s potential to inhibit HBV replication in cells.
7. Outlook

The HBV RNaseH is a promising but unexploited target for antiviral drug development. Anti-RNaseH drugs are envisioned to be used in combination with the existing nucleos(t)ide analogs to suppress HBV replication much further than is currently possible. The goal will be to suppress viral replication enough to block replenishment of the cccDNA in the liver, permitting turnover of the cccDNA and elimination of infected cells by the immune system to clear the infection. The long apparent halflife of the cccDNA implies that therapy will be prolonged and may well benefit from co-administration with novel immune-stimulating therapies that are under development (Bertoletti and Gehring, 2013 and Gehring et al., 2014). Therefore, minimizing toxicity and optimizing specificity and pharmacokinetic properties of potential RNaseH drugs will be especially important.
Acknowledgements

Writing of this review was supported in part by R01 AI104494 to J.T. and an ancillary study within the NIDDK-sponsored Hepatitis B Virus Research Network (U01 DK082871 to Adrian Di Bisceglie and J.T.). We thank our collaborators Drs. Stuart Le Grice, Ryan Murelli, Fabrice Bailly, Philippe Cotelle, and Marvin Meyers for their many contributions to this RNaseH project. Use of some of these compounds against HBV is covered by U.S. Patent Application 13/072201 (Pending).

StephenW

超过190个化合物的αHT，N-hydroxyisoquinolinedione，napthyridinone，dioxobutanoic酸，hydroxychromenone，Elvitegravir，雷特格韦，aminocyanothiophene，hydroxyxanthenone，cyanopyran和thienopyrimidinone化学家庭已筛选出对HBV RNA酶H。十九化合物抑制乙肝病毒的RNA酶HμM⩽20，具有低微摩尔的IC50值的最佳命中[（Cai等，2014年，胡适等人，2013年，鲁等人，2015年和塔维斯等人，2013年）和未发表的。强命中αHTs，N-hydroxyisoquinolinediones，napthyridinones和hydroxychomenones中进行观察。在所有情况下，该化合物对HBV基因型C，D和/或H.类似活动的抑制剂似乎通过螯合在RNA酶H活性部位的二价阳离子，因为除去的三个路易斯碱部分的一种或改变其角度相对于行动对方烧蚀抑制[（Cai等，2014年和Lu等，2015）和未发表的。反筛选抗人RNaseH1表明，大多数这些筛选的命中也积极对人酶，但在这两种酶的敏感性的差异是明显的（Lu等人，2015）。这表明，越来越多的选择性为乙肝病毒酶必须是在化学优化的一大重点。

三十七个化合物已经过测试，抑制文化HBV复制的能力; 10抑制HBV的复制⩽20μM（表1）。病毒正极性DNA链的优先抑制证实抑制是由于阻断了所有化合物的病毒RNA酶H活性除了＃56。观察到温和的细胞毒性对于所有活性化合物，具有CC 50值范围为6.1至79微米。因此，这将是必要的RNA酶H抑制剂的开发过程中减少的细胞毒性。对大多数这些抑制剂的EC 50值是比它们的IC 50值低得多。对此的原因定量差异是未知的，但它可能是由于在细胞中的化合物的积累，代谢抑制剂，以更积极的方式，或最可能的，该重组RNA酶H不重述其天然的酶的所有方面上下文全长HBV聚合酶蛋白的一部分。尽管如此，该重组酶的强抑制是的化合物的潜在抑制乙型肝炎病毒复制的细胞中一个极好的预测。
7.展望

乙肝病毒RNA酶H是一种很有前途，但尚未开发的目标，抗病毒药物开发。抗RNA酶H药物计划要组合使用现有的核苷（酸）类似物到远远超出目前可能抑制HBV复制。的目标将是抑制病毒复制，足以阻断cccDNA的补给，在肝脏，允许成交的cccDNA和消除感染细胞的免疫系统，以清除感染。该cccDNA的长半衰期明显暗示治疗将持续很长时间，并可能从共同给药与新型免疫刺激疗法，正在发展中受益（BERTOLETTI和Gehring集团，2013年和Gehring等人，2014年）。因此，最大限度地减少毒性及优化潜在RNA酶H的药物的特异性和药动学性质将是特别重要的。
致谢

这次审查的是写作部分由R01 AI104494到JT支持和NIDDK赞助乙型肝炎病毒研究网络（U01 DK082871阿德里安迪比谢列和JT）内的配套研究。我们感谢我们的合作者博士。斯图尔特·格莱斯，瑞安Murelli，法布里斯贝利，菲利普Cotelle，和马文迈耶斯他们为这个项目RNA酶H许多贡献。使用一些抗HBV这些化合物已获得美国专利申请13/072201（待定）。

StephenW

全文:
http://www.sciencedirect.com/sci ... i/S0166354215000820

9病成医

一个开发抗病毒药物的新思路？远水不解近渴。