环庚三烯酚酮羟基化物的衍生物对抗病毒新靶点HBV RNAseH的作用机制研究

Hepatitis B Virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. Therapy primarily employs nucleos(t)ide analogs that suppress viral DNA synthesis by the viral reverse transcriptase very well but rarely cure the infection, therefore, novel drugs against targets other than the reverse transcriptase activity are urgently needed. Reverse transcription requires the viral ribonuclease H (RNAseH) to destroy the viral RNA after it has been copied into DNA. Therefore inhibiting the HBV RNAseH would block both cccDNA replenishment and secretion of new virions. Consequently, RNAseH inhibitors could be used to augment the existing nucleos(t)ide analog drugs to improve suppression of the cccDNA. Previously, we had discovered β-thujaplicinol - a hydroxylated tropolone derivative isolated from the heartwood of Western Red Cedar trees that inhibits the HBV RNAseH with a 50% inhibitory activity of 5.9 ± 0.7 μM, It blocked replication of HBV genotypes A and D in culture by inhibiting the RNAseH activity with an estimated EC50 of 5 μM and a CC50 of 10.1 ± 1.7 μM. In this research, we would investigate the mechanism of hydroxylated tropolone derivative on the HBV RNAseH: first, we would screen the potential HBV RNAseH inhibitors based on a low throughput screening pipeline. then analysis whether these inhibitors would affect HBV reverse transcription such pgRNA encapsidation, the minus strand DNA initiation 、elongation, RNA primer translocation or elongation of plus strand DNA. In addition, we would analysis the inhibition for synthesis of cccDNA which is the key factor of HBV infection. Finally, we would evaluate the efficacy of inhibition by the transgenic mice. This research will reveal how the hydroxylated tropolone derivative affect HBV replication and cccDNA systhesis by blocking the activity of HBV RNAseH, in addition, this would provide essential guidance for developing anti HBV drugs for thses inhibitors.

筛选新的抗HBV药物及机制研究是国际研究的重点，前期研究中我们发现环庚三烯酚酮羟基化物的衍生物－β-thujaplicinol能作用HBV RNAseH而抑制病毒复制。本项目将利用前期建立的体外biochemical和cell culture药敏体系，从17种该类衍生物中筛选出几种能作用于HBV RNAseH而抑制病毒复制的抑制剂，围绕HBV RNAseH在HBV反转录复制中的作用，通过primer extension analysis等策略逐步弄清楚该类抑制剂在HBV反转录复制环节和cccDNA合成环节中的作用，解析其作用机制，并通过HBV转基因小鼠模型在体内评价该类药物对HBV复制及肝组织学的作用效应，其研究结果不仅阐明了环庚三烯酚酮羟基化物的衍生物中对HBV RNAseH的作用机制，同时通过体外和体内实验评价了该类药物对HBV RNAseH的抑制效应，为该类药物的研发提供了科学依据。

乙型肝炎病毒（Hepatitis B virus，HBV）是一种嗜肝DNA病毒，引起急性和慢性感染。现有慢乙肝治疗药物（干扰素和核苷酸类似物）由于应答较低或耐药问题的存在，因此寻找新的抗病毒药物或宿主因子作为新靶点来治疗慢乙肝患者是目前乙肝治疗中最重要的问题。本课题一方面从抗病毒新靶点HBV RNAseH出发，发现化合物46（β-thujaplicinol）在体外和细胞模型上能够抑制HBV RNAseH，从而抑制病毒复制，为该类化合物的研发提供了基础；另一方面我们解析了宿主限制性因子APOBEC3B和SAMHD1与病毒相互作用的机制，为抗病毒提供了新的策略。 .(1) HBV RNAseH抑制剂的筛选及机制研究: 我们首先利用基于体外筛选HBV RNAseH抑制剂的体系，从17种环庚三烯酚酮羟基化物的衍生物中首次筛选出能够作用于HBV RNAseH的化合物46（β-thujaplicinol）在体外能有效抑制HBV RNAseH活性，体外半数抑制常数IC50 = 5.9uM，对human RNAseH没有明显的抑制作用；在HBV转染-复制的细胞模型上阐明β-thujaplicinol通过阻滞病毒正链DNA的合成来抑制病毒复制的分子机制，为该类药物的研发提供了基础。.(2) 宿主限制性因子APOBEC3B和SAMDH1与病毒相互作用的机制: 从APOBEC3s家族出发，我们不仅揭示了HBV感染的HCC中，cccDNA表达水平的降低可能与肿瘤组织中APOBEC3B的高表达相关，并首次阐明APOBEC3B蛋白与病毒核心蛋白相互作用，对病毒负链DNA进行编辑作用的分子机制；从SAMHD1因子出发，我们不仅揭示了CDK2激酶磷酸化SAMHD1，负调控其抗病毒的分子机制，并筛选出通过阻止SAMHD1磷酸化而抑制病毒复制的CDK2激酶抑制剂II，为该类药物的研发提供了基础。. 本项目不仅筛选和鉴定β-thujaplicinol作用HBV RNAseH，抑制HBV复制的的分子机制，同时从宿主重要的限制性因子APOBEC3B和SAMHD1出发，阐明了宿主与病毒的相互作用机制，为抗病毒疗法提供新靶点和新思路。. 项目一共发表文章11篇，其中SCI 5篇，中文核心期刊6篇，培养研究生4名，研究结果分别在2016年国际HBV分子病毒学等国内外会议进行交流。