靶向流感病毒进入(细胞)抑制剂研究进展

目前临床上抗流感病毒感染的药物主要有M2离子通道抑制剂及神经氨酸酶抑制剂两大类.随着流行性感冒、季节性流感、高致病性禽流感的爆发以及耐药流感病毒株的出现,使得对具有新作用机制的抗流感药物的需求更加紧迫.近年来,随着流感病毒进入细胞分子机制研究的深入,为以"病毒进入细胞为靶"的抗流感抑制剂的研究带来了契机.本文在介绍流感病毒进入细胞分子机制的基础上,重点介绍了靶向流感病毒进入的抑制剂及其作用机制的最新研究进展.     

紫草萘醌类化合物对乳酸脱氢酶和乙醇脱氢酶的共价修 饰作用

为研究紫草萘醌类化合物的细胞毒性作用机制,从新疆软紫草根中分离出四种紫草萘醌类化合物β,β-二甲基丙烯酰阿卡宁(1),乙酰阿卡宁(2),β-乙酰氧基异戊酰阿卡宁(3)和阿卡宁(4)。研究了四种天然紫草萘醌类化合物对乳酸脱氢酶和乙醇脱氢酶的共价修饰作用。酶活力测定结果表明,这四种紫草萘醌类化合物对两种脱氢酶都具有不同程度的抑制作用;酶分子中游离氨基和巯基修饰率的测定结果表明,紫草萘醌类化合物对两种酶的抑制作用主要是通过与酶分子中的巯基共价结合产生的。     

一枝蒿酮酸酰胺衍生物的合成和抗A3，B型流感病毒和单纯Ⅰ，Ⅱ型疱疹病毒活性研究

从新疆一枝蒿巾分离得到了单体化合物一枝蒿酮酸,然后以一枝蒿酮酸和有机胺为原料,存偶联剂DCC,HOBt/DMAP的作用下,合成了13种未见文献报道的一枝蒿酮酸酰胺衍生物2a~2m.所合成的化合物经过IR,1HNMR,ESI-MS等分析方法进行了表征,并对化合物2a~2m进行初步的体外抗A3,B犁流感病毒和单纯Ⅰ,Ⅱ型疱疹病毒活性研究.初步试验结果表明化合物2a同时具有抗A3,B型流感病毒活性,而且抗B型流感病毒活性比母体化合物的活性较高.化合物2d的抗B犁流感病毒活性比母体化合物高16倍,化合物2e同时具有较强的抗单纯Ⅰ,Ⅱ型疱疹病毒活性.     

一枝蒿酮酸酰胺衍生物的合成和抗A3, B型流感病毒和 单纯I, II型疱疹病毒活性研究

从新疆一枝蒿中分离得到了单体化合物一枝蒿酮酸, 然后以一枝蒿酮酸和有机胺为原料, 在偶联剂DCC, HOBt/DMAP的作用下, 合成了13种未见文献报道的一枝蒿酮酸酰胺衍生物2a～2m. 所合成的化合物经过IR, 1H NMR, ESI-MS等分析方法进行了表征, 并对化合物2a～2m进行初步的体外抗A3, B型流感病毒和单纯I, II型疱疹病毒活性研究. 初步试验结果表明化合物2a同时具有抗A3, B型流感病毒活性, 而且抗B型流感病毒活性比母体化合物的活性较高. 化合物2d的抗B型流感病毒活性比母体化合物高16倍, 化合物2e同时具有较强的抗单纯I, II型疱疹病毒活性.     

桂皮酸糖酯类衍生物的合成

桂皮酸作为常用的食用香料、新鲜水果和蔬菜等的防腐剂,是一种来源广泛,对人体安全的物质,被美国列为GPAS范围.有报道把它称为一种调节植物分化和生长的激素,还具有抗微生物、抗炎、抗血小板凝聚的作用[1].此外,一系列研究表明桂皮酸及其衍生物对黑色素瘤细胞、结肠癌细胞、流感病毒等多种肿瘤细胞和病毒具有抑制作用[2-6].但由于桂皮酸分子中含有羧基,对胃肠道有较强的刺激作用,因此,对桂皮酸的结构修饰已有研究报道[7].     

酚化合物对胰岛素淀粉样纤维化的抑制作用

采用牛胰岛素作为模型多肽分子, 对几种结构相近的简单多酚的抗多肽淀粉样纤维化作用进行了研究. 结果表明, 邻苯二酚和对苯二酚对胰岛素纤维化具有抑制作用, 并通过形成醌中间体对多肽链进行修饰, 与对苯醌作用类似; 而苯酚和间二苯酚在相同条件下, 既不能修饰多肽也无抑制纤维化作用. 在无氧条件下, 邻苯二酚和对苯二酚对胰岛素纤维化的抑制作用明显降低, 说明酚化合物经氧化形成的醌中间体是其抗胰岛素纤维化的主要活性结构.     

血必净抗炎作用药效物质基础和多靶点作用效应

研究了血必净抗炎作用的药效物质基础, 并在分子层面上对复方多靶点作用进行阐释. 借助于计算机辅助药物设计技术, 构建血必净化学成分数据库, 综合应用同源模建、分子对接、药效团模型、数据库搜索等方法, 探讨其与炎症靶点5-脂氧合酶(5-LOX)、环氧合酶-2(COX-2)、IKK-2受体的相互作用关系. 血必净化学成分中与靶点5-LOX、COX-2、IKK-2结合效应较好的分别有30、36、8个分子; 有16个分子与2个或3个靶点存在作用, 其中15个分子对靶点5-LOX和COX-2有抑制作用, 迷迭香酸对3个靶点均有作用. 从分子层次上阐释了复方血必净抗炎作用的药效物质基础和多靶点作用效应, 为血必净复方的临床应用提供了科学依据; 同时, 也为寻找新型抗炎药物提供一定的参考和借鉴.     

新型一枝蒿酮酸异噁唑衍生物的合成及其抗A,B型流感病毒活性研究

为了提高一枝蒿酮酸的生物活性,以一枝蒿酮酸和3-取代苯基-5-氨甲基-异噁唑为原料,在偶合试剂DCC,HOBt/DMAP的作用下,合成了6个未见文献报道的含异噁唑的一枝蒿酮酸酰胺衍生物3a～3f.所合成的化合物均经过IR,1H NMR,13C NMR,ESI-MS等分析方法表征及初步体外抗A(H3N2,H1N1)型和B型流感病毒活性研究.初步实验结果表明:化合物3c同时具有抗A(H3N2)型和B型流感病毒活性,化合物3c和3e表现出比母体化合物强的抗B型流感病毒活性.     

基于计算机模拟技术对JAK2抑制剂的定量构效关系、分子设计和分子动力学研究

本文选取了52个对Janus激酶2(JAK2)有抑制作用的小分子化合物,分别使用3D-QSAR中的CoMFA和CoMSIA方法构建了两个可靠的、具有预测能力的模型,并利用分子对接分析数据集化合物与JAK2蛋白的相互作用,表明化合物主要通过氢键和范德华作用与JAK2靶蛋白结合。根据3D-QSAR模型的分析结果,设计了40个化合物,利用构建的模型预测其抑制活性;使用软件预测了化合物的药代动力学(ADME)参数,开展分子对接模拟,最终选择化合物D01和D22与JAK2靶蛋白进行了分子动力学模拟研究,结果显示两个复合物结合构象稳定,与分子对接结果趋势一致。本研究的结果可以为JAK2抑制剂的研发提供一些新的思路,为临床开发此类药物提供理论支撑。     

五环三萜葡萄糖缀合物的设计、合成及体外抗流感病毒活性研究

前期研究发现齐墩果酸(OA)的C28位葡萄糖偶联物在细胞水平具有显著抗流感病毒的活性,但不同五环三萜母核偶联葡萄糖抗流感病毒活性的影响并未阐明.本研究采用不同五环三萜母核,通过Cu(I)催化的炔烃-叠氮化物环加成反应(Cu AAC),与葡萄糖偶联制备得到化合物物4c～8d,通过体外抗流感病毒A/WSN/33(H1N1)活性研究,发现化合物4d具有显著的抗流感病毒活性(IC₅₀为9.09μmol/L),且对MDCK(Madin-Darbey CanineKidney)细胞无明显毒性(CC50>100μmol/L).构效关系研究明确了葡萄糖模块以及五环三萜母核对化合物抗流感病毒活性的提升均具有重要作用.本研究进一步完善了五环三萜及其衍生物抗流感病毒的构效关系,为这类天然产物抗病毒的深入研究奠定了基础.     

A negative stain for electron microscopic tomography

While negative staining can provide detailed, two-dimensional images of biological structures, the potential of combining tomography with negative staining to provide three-dimensional views has yet to be fully realized. Basic requirements of a negative stain for tomography are that the density and atomic number of the stain are optimal, and that the stain does not degrade or rearrange with the intensive electron dose (~10? e/nm2) needed to collect a full set of tomographic images. A commercially available, tungsten-based stain appears to satisfy these prerequisites. Comparison of the surface structure of negatively stained influenza A virus with previous structural results served to evaluate this negative stain. The combination of many projections of the same structure yielded detailed images of single proteins on the viral surface. Corresponding surface renderings are a good fit to images of the viral surface derived from cryomicroscopy as well as to the shapes of crystallized surface proteins. Negative stain tomography with the appropriate stain yields detailed images of individual molecules in their normal setting on the surface of the influenza A virus.     

Pharmacophore modelling,virtual screening and molecular docking studies on PLD1 inhibitors

Lipid metabolism plays a significant role in influenza virus replication and subsequent infection. The regulatory mechanism governing lipid metabolism and viral replication is not properly understood to date, but both Phospholipase D (PLD1 and PLD2) activities are stimulated in viral infection. In vitro studies indicate that chemical inhibition of PLD1 delays viral entry and reduction of viral loads. The current study reports a three-dimensional pharmacophore model based on 35 known PLD1 inhibitors. A sub-set of 25 compounds was selected as the training set and the remaining 10 compounds were kept in the test set. One hundred and twelve pharmacophore models were generated; a six-featured pharmacophore model (AADDHR.57) with survival score (2.69) produced a statistically significant three-dimensional quantitative structure–activity relationship model with r² = 0.97 (internal training set), r² = 0.71 (internal test set) and Q² = 0.64. The predictive power of the pharmacophore model was validated with an external test set (r² = 0.73) and a systematic virtual screening work-flow was employed showing an enrichment factor of 23.68 at the top 2% of the dataset (active and decoys). Finally, the model was used for screening of the filtered PubChem database to fetch molecules which can be proposed as potential PLD1 inhibitors for blocking influenza infection.     

Inhibition of influenza virus sialidase and anti-influenza virus activity by plant flavonoids

Flavonoids (103 species) were tested for inhibitory activity against influenza virus sialidase using sodium p-nitrophenyl-N-acetyl-alpha-D-neuraminate as substrate. 5,7,4'-Trihydroxy-8-methoxyflavone from the root of Scutellaria baicalensis showed the most potent activity (IC50, 55 microM), and this flavone appeared to be a non-competitive inhibitor of the enzyme. Whereas, negligible or weak inhibitory activities were observed for mouse liver sialidase, beta-galactosidase and alpha-mannosidase as tested. This flavone also inhibited the infection by influenza virus A/PR/8/34 of Madin-Darby canine kidney cells, and replication of the virus in the allantoic sack of embryonated egg. These results suggest that flavone, which has potent influenza virus sialidase inhibitory activity, may have anti-influenza virus activity.     

Proximity Ligation‐Based Fluorogenic Imaging Agents for Neuraminidases

Reagents to visualize and localize neuraminidase activity would be valuable probes to study the role of neuraminidases in normal cellular processes as well as during viral infections or cancer development. Herein, a new class of neuraminidase‐imaging probes that function as proximity ligation reagents by releasing a highly reactive fluorophore that tags nearby cellular material is described. It is further demonstrated that it is possible to create an influenza virus‐specific reagent, which can specifically detect influenza virus infections in mammalian cells. These reagents have potential use as specific histological probes independent of viral antigenicity and, therefore, offer some advantages over commonly used anti‐neuraminidase antibodies.     

Predicting interspecies transmission of avian influenza virus based on wavelet packet decomposition

Using wavelet packet decomposition, the energy coefficients in the fifth level of viral protein sequences were achieved to predict interspecies transmission. Since avian-origin influenza viruses could have high sequence similarities with human-origin avian influenza virus and could have the phenotype of interspecies transmission, viral data should be filtered to prevent the misconduct of feature selection and false performance of predicting models. Considering the balance of data size, the empirical cut-off value 97% was used to screen avian-origin influenza virus with high sequence similarity. The excellent performances of cross validation show that the SVM model has the best capability of predicting transmission and evaluating the contribution of five amino acid factors. The robust model was finally used to evaluate the filtered data of avian-origin virus and the results confirmed that double check for ambiguous phenotype of avian-origin virus with high sequence similarity was necessary and part of them have the ability to across species barriers.     

Detection of intact influenza viruses using biotinylated biantennary S-sialosides

We report the modular synthesis of robust, biotinylated biantennary sialylglycoconjugates and their ability to differentiate between two type A influenza strains. This is the first demonstration of glycoconjugate-based discriminatory capture and detection of two strains of intact influenza virus, in the presence of the innate enzymatic activity of viral neuraminidases. We also demonstrate a "carboassay" using glycoconjugates as capture and reporter elements, which therefore, does not require antibodies. The capture of intact influenza viruses is of potential benefit for clinical diagnostics.     

Antiviral properties of cage compounds. New prospects

The published data of the last 15 years on the antiviral activity and the mechanism of action of cage compounds are integrated and described systematically. The considerable interest in the cage compounds as antiviral agents is related to the specific features of the spatial structure of this class of derivatives and high lipophilicity and rigidity of the carbon cage, which allows these molecules to easily penetrate through the lipid layer of biological membranes. Data on the ion channel structure of influenza A and hepatitis C viruses and docking data for some cage structures to these channels are presented. Data on the antiviral properties of cage compounds against RNA genome viruses, the influenza A virus and its mutant strains, hepatitis C virus, human immunodeficiency viruses, and other RNA-containing viruses, are presented. The efficiency of cage compounds against the DNA-genome viruses, herpes virus, cytomegalovirus and orthopoxviruses, is demonstrated. The proven participation of aminoadamantanes in the suppression of early stages of the influenza virus life cycle suggests that efficient inhibitors of not only the influenza virus but also other RNA- and DNA-containing viruses could be found among the cage molecules.     

A network-based pharmacology study of active compounds and targets of Fritillaria thunbergii against influenza

Seasonal and pandemic influenza infections are serious threats to public health and the global economy. Since antigenic drift reduces the effectiveness of conventional therapies against the virus, herbal medicine has been proposed as an alternative. Fritillaria thunbergii (FT) have been traditionally used to treat airway inflammatory diseases such as coughs, bronchitis, pneumonia, and fever-based illnesses. Herein, we used a network pharmacology-based strategy to predict potential compounds from Fritillaria thunbergii (FT), target genes, and cellular pathways to better combat influenza and influenza-associated diseases. We identified five compounds, and 47 target genes using a compound-target network (C-T). Two compounds (beta-sitosterol and pelargonidin) and nine target genes (BCL2, CASP3, HSP90AA1, ICAM1, JUN, NOS2, PPARG, PTGS1, PTGS2) were identified using a compound-influenza disease target network (C-D). Protein-protein interaction (PPI) network was constructed and we identified eight proteins from nine target genes formed a network. The compound-disease-pathway network (C-D-P) revealed three classes of pathways linked to influenza: cancer, viral diseases, and inflammation. Taken together, our systems biology data from C-T, C-D, PPI and C-D-P networks predicted potent compounds from FT and new therapeutic targets and pathways involved in influenza.     

抗流感病毒抑制剂Nucleozin衍生物的设计合成及抗病毒活性评价

小分子化合物Nucleozin作为靶向流感病毒核蛋白的抑制剂具有良好的抑制活性。本文围绕Nucleozin分子中与哌嗪环直接相连的芳环部分进行研究。通过钯催化偶联反应合成了一系列Nucleozin衍生物,通过检测所合成化合物对流感病毒H1N1的抑制活性,明确了Nucleozin分子中该部分的构效关系。利用甲基在药物分子设计中的作用,设计将分子中的氯原子替换为甲基,发现与原型分子Nucleozin相比其抑制活性有了明显的提高。本文的结果对该类分子成药性的提高具有积极意义。     

PHOTOINACTIVATION OF INFLUENZA VIRUS FUSION AND INFECTIVITY BY ROSE BENGAL

Rose bengal inactivated influenza virus upon exposure to light. Infectivity and fusion were inactivated with the same dose dependence, supporting the suggestion that the virucidal activity of photodynamic agents against enveloped viruses may be generally due to inactivation of their fusion protein(s). Concentrations required for inac-ti vation were found to depend upon the ratio of rose bengal to virus, rather than on the nominal aqueous concentration. Fusion-competent virosomes were inactivated similarly to intact virus particles. The HA_Z portion of the influenza fusion protein HA underwent two different, apparently mutually exclusive modifications upon illumination with rose bengal: cross-linking, and conversion to a form that moved slightly more slowly on sodium dodecyl sulfate poly-acrylamide gel electrophoresis. Inactivation of viral fusion was inhibited by oxygen removal or addition of azide or β-carotene, and was enhanced by D₂O, consistent with partial involvement of singlet oxygen. The possibility of a second mechanism of viral photoinactivation, by direct interaction between the viral fusion protein and the pho-toactivated dye, is also discussed.