新型抗乙型肝炎病毒化合物的活性评价及机制探索

发布时间：2018-04-18 21:31

本文选题：乙型肝炎病毒 + 哒嗪酮类化合物　；参考：《中国科学院上海药物研究所》2016年博士论文

【摘要】：慢性乙型肝炎病毒(HBV)感染是严重的传染性疾病,由于目前临床上用于抗HBV的药物仅有免疫调节类和核苷类药物两类,而这两类药物又均存在一定的局限性,且无法彻底清除患者体内的病毒,该领域的研究者们一直致力于新型非核苷类抗HBV化合物的探索工作。本课题首次报道了哒嗪酮类化合物具有显著的抗HBV活性。其中的代表性化合物3711,可有效降低细胞内及细胞培养上清中的HBV DNA水平,半数抑制浓度分别为1.5±0.2μM及1.9±0.1μM,治疗窗较大。在3711的机制探索实验中,我们发现3711给药可诱导细胞内产生不含病毒核酸的“空”病毒颗粒,HBV病毒颗粒形态无改变,但病毒颗粒在CsCl密度梯度离心实验中的沉降速率以及在1.8%的非变性琼脂糖凝胶中的电泳移动速率在给药后均发生改变。3711诱导产生的“空”病毒核心颗粒可直接以“裸”核心颗粒形式分泌出胞,也可包膜后以包膜病毒颗粒形式分泌出胞,二者由于缺少病毒基因组均无法进行新一轮的感染扩增。另外,3711为新型非核苷类小分子化合物,其抗病毒活性与HBV聚合酶无关,因此可有效抑制核苷类药物耐药病毒突变株的复制。3711的活性仅与HBV核心蛋白1-144位氨基酸,即组装结构域相关。构建将HBV核心蛋白124位缬氨酸突变为具有较大疏水侧链的色氨酸突变体(V124W),增加核心蛋白亚基之间的疏水作用,可模拟3711的活性,证明3711可能通过作用于核心蛋白亚基之间的疏水口袋干扰HBV核壳体包装过程,诱导“空”核心颗粒组装,从而发挥抗HBV活性。为进一步获取高效低毒的抗病毒化合物,本课题组与上海药物研究所胡有洪课题组合作,对哒嗪酮类小分子化合物进行结构优化及改造,目前已经获得溶解度得到大幅提高的化合物5045,以及抗HBV活性得到明显改善的化合物5165及5174。此外,我们将3711与正在进行临床I期实验的抗HBV新药异噻氟定(NZ-4)的作用机制进行比较,进一步验证了尽管二者均通过诱导细胞内产生不含病毒核酸的“空”病毒颗粒发挥抗病毒活性,但其作用靶点及分子机制不同,NZ-4的活性依赖于HBV核心蛋白C末端第一精氨酸结构域的存在,而3711的抗病毒活性仅与核心蛋白N端的疏水口袋相关。综上所述,本课题完成了对两类非核苷类小分子化合物抗HBV复制的机制研究。首次发现了一类哒嗪酮小分子化合物具有良好的抗HBV活性,其通过作用于HBV核壳体组装过程发挥作用,不仅可作为工具药来探索HBV核壳体组装过程,也为未来慢性乙型肝炎患者的治疗提供了更多的可能性。其次,明确了异噻氟定(NZ-4)与目前报道的所有抗病毒化合物的作用机制均不同,是目前唯一一个依赖于HBV核心蛋白C末端第一精氨酸富集区发挥抗HBV活性的化合物,并已经进入临床研究阶段,其作用机制的独特性使其可能为未来慢性乙型肝炎的治愈以及新型药物的研发提供了一个新的选择。
[Abstract]:Chronic hepatitis B virus (HBV) infection is a serious infectious disease. At present, there are only two kinds of drugs, immunomodulatory drugs and nucleoside drugs, which have some limitations.And unable to completely eliminate the virus in patients, researchers in the field have been working on new non-nucleoside anti-HBV compounds.In this paper, we report for the first time that pyridazinones have remarkable anti-HBV activity.The representative compound 3711 could effectively reduce the level of HBV DNA in cells and supernatants of cell culture. The median inhibitory concentrations were 1.5 卤0.2 渭 M and 1.9 卤0.1 渭 M, respectively.In the mechanism exploration experiment of 3711, we found that 3711 administration could induce the formation of "empty" virus particles without virus nucleic acid in the cells, and the morphology of HBV virus particles remained unchanged.However, the sedimentation rate of virus particles in CsCl density gradient centrifugation test and the electrophoretic mobility rate in 1.8% non-denatured agarose gel changed after administration. 3711-induced "empty" virus core particles can be directly changed by"naked" core granules secrete cells,They can also secrete cells in the form of encapsulated virus particles, and neither of them can carry out a new round of infection amplification due to the lack of virus genome.In addition, Y3711 is a new type of non-nucleoside small molecule compound, its antiviral activity is independent of HBV polymerase, so it can effectively inhibit the replication of nucleoside drug-resistant virus mutant. 3711 is only 1-144 amino acids of HBV core protein.That is, the assembly domain is related.The HBV core protein 124-valine mutant was constructed and transformed into a tryptophan mutant with a large hydrophobic side chain, which can enhance the hydrophobic interaction between the core protein subunits and mimic the activity of 3711.It is proved that 3711 may interfere with the packaging process of HBV nuclear shell by acting on the hydrophobic pocket between the core subunits and induce the assembly of "empty" core particles, thus exerting anti- activity.In order to obtain antiviral compounds with high efficiency and low toxicity, our research group, in cooperation with Hu Youhong group of Shanghai Institute of Pharmacology, optimized and modified the structure of pyridazinone small molecular compounds.Up to now, 5045 compounds with significantly improved solubility and 5165 and 5174 compounds with significantly improved anti- HBV activity have been obtained.In addition, we compared the action mechanism of 3711 with that of Isotiafuridine (NZ-4), a new anti- HBV drug, which is currently undergoing phase I clinical trials.It is further verified that although both of them play antiviral activities by inducing the production of "empty" viral particles containing virus-free nucleic acids in cells,However, the activity of NZ-4 depends on the existence of the first arginine domain at the C-terminal of HBV core protein, and the antiviral activity of 3711 is only related to the hydrophobic pocket of the N-terminal core protein.In conclusion, the mechanism of anti-HBV replication of two kinds of non-nucleoside small molecular compounds has been studied.For the first time, a class of pyridazinone small molecular compounds have been found to have good anti- HBV activity, which can not only be used as a tool to explore the HBV nuclear shell assembly process, but also play an important role in the HBV nuclear shell assembly process.It also provides more possibilities for the treatment of chronic hepatitis B patients in the future.Secondly, it is clear that the mechanism of isothiafuridine NZ-4 is different from that of all the antiviral compounds reported at present, and it is the only one that relies on the first arginine rich region at the C-terminal of HBV core protein to exert anti- activity.And it has entered the stage of clinical research. The unique mechanism of its action may provide a new choice for the cure of chronic hepatitis B and the development of new drugs in the future.
【学位授予单位】：中国科学院上海药物研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

【相似文献】