研究报告：槲皮苷： 一种抑制钙调蛋白磷酸酶（CN）的新型免疫抑制剂

目的以钙调蛋白磷酸酶(CN)为靶酶,从中草药中寻找高效、低毒的免疫抑制剂。方法以CN为靶点,筛选并分离能够抑制其活性的天然化合物。在细胞和动物水平评价该化合物的免疫抑制效果及毒副作用,并通过荧光猝灭、分子对接、免疫印迹、双荧光素酶报告基因、实时定量PCR等实验探究天然化合物与CN的作用机理及可能的免疫抑制作用机制。结果槲皮苷在体外和细胞内均能抑制CN的活性,并且能够有效抑制小鼠脾细胞的增殖,缓解小鼠迟发超敏反应。毒理研究结果显示,槲皮苷对实验小鼠无急性毒性,且毒副作用很小。荧光猝灭实验和分子对接分析表明,槲皮苷可能与CN上的两个位点相结合,并通过CN-NFAT通路参与调节免疫反应。结论通过筛选获得了新型CN抑制剂槲皮苷。槲皮苷具有成为新型低毒免疫抑制剂的潜在可能。     

天然小分子化合物调节小胶质细胞预防阿尔茨海默病免疫失衡（英文）

阿尔茨海默病(Alzheimer's disease,AD)是最常见的中枢神经系统退行性疾病,其发病机制复杂,至今仍未完全阐明.最近的研究表明,小胶质细胞过度激活、炎症因子的过度产生与AD的发病密切相关.小胶质细胞受体及下游通路的异常调节可导致AD患者及AD实验动物的免疫失衡.天然小分子化合物通过激活小胶质细胞的抑炎受体,抑制促炎受体或调节Aβ清除受体,可逆转免疫失衡.本文综述了小胶质细胞在AD慢性炎症中的作用机制,并总结天然小分子化合物通过调节小胶质细胞受体及其下游通路在AD免疫稳态中的有益作用.     

天然小分子化合物调节小胶质细胞预防阿尔茨海默病免疫失衡*

阿尔茨海默病(Alzheimer"s disease, AD)是最常见的中枢神经系统退行性疾病，其发病机制复杂，至今仍未完全阐明. 最近的研究表明，小胶质细胞过度激活、炎症因子的过度产生与AD的发病密切相关. 小胶质细胞受体及下游通路的异常调节可导致AD患者及AD实验动物的免疫失衡. 天然小分子化合物通过激活小胶质细胞的抑炎受体，抑制促炎受体，或调节Aβ清除受体，可逆转免疫失衡. 本文综述了小胶质细胞在AD慢性炎症中的作用机制，并总结天然小分子化合物通过调节小胶质细胞受体及其下游通路在AD免疫稳态中的有益作用.     

天然小分子诱生机体抗病毒细胞因子研究

天然小分子是中药、药用植物和天然药物的重要活性成分,是重要的药物资源研究方向和内容。大多数抗病毒药用植物,如夏枯草、黄芪和黄芩等,其活性成分均为天然小分子。抗病毒细胞因子为一类生物体中具有联系机体固有免疫和特异性免疫应答,捕杀或抑制体内病毒的小分子功能蛋白。近年来研究表明,植物中的多酚类、苷类以及寡糖等小分子化合物可调控机体内源抗病毒细胞因子的表达水平,继而作用于各类DNA或RNA病毒:一方面刺激机体产生抗病毒蛋白,直接捕杀病毒;另一方面联动机体固有免疫和获得性免疫应答,抑制病毒复制,抗病毒感染,清除被病毒感染的细胞。本文综述了近几十年药用植物天然小分子诱生机体细胞因子抗病毒的作用及机制研究,并由此提出这类活性天然小分子将可能成为新一类的抗病毒药物。     

植物来源药用天然产物的合成生物学研究进展

大多数药用天然产物在植物中含量低微,提取分离困难;而且这些化合物一般结构复杂,化学合成难度大,还容易造成环境污染。基于合成生物学技术获得药用天然产物具有绿色环保和可持续发展等优点。文中以药用萜类化合物人参皂苷、紫杉醇、青蒿素、丹参酮,生物碱类化合物长春新碱、吗啡以及黄酮类化合物灯盏花素为例,总结了植物来源药用萜类、生物碱类和黄酮类化合物的生物合成途径及合成生物学研究进展,介绍了药用天然产物合成生物学研究的关键技术与方法,并展望了合成生物学技术在药用天然产物研究与开发方面的应用前景。     

高亲和力抗乙型肝炎病毒PreS1的人源单链抗体的获得:天然及免疫抗体库的对比研究( 英文)

以健康人的外周血淋巴细胞为来源 ,以偶联BSA的乙型肝炎病毒PreS1肽体外免疫 .分别从免疫和未经免疫的淋巴细胞提取RNA ,扩增抗体基因 ,构建大容量天然单链抗体 (scFv)噬菌体展示文库和体外免疫scFv抗体库 .以PreS1肽进行 3轮淘选后 ,抗原抗体反应结果显示 ,从免疫库中获得了亲和力 10 -7～ 10 -8M的抗乙型肝炎病毒PreS1的单链抗体 ,高于天然库的结果 (10 -6～ 10 -7M ) .测序结果表明两株抗体均为人抗体 .为基因工程抗体用于临床治疗乙型肝炎奠定基础 .同时证明淋巴细胞体外免疫方法构建的免疫抗体库优于大容量天然抗体库 .     

高亲和力抗乙型肝炎病毒PreS1的人源单链抗体的获得:天然及免疫抗体库的对比研究

以健康人的外周血淋巴细胞为来源,以偶联BSA的乙型肝炎病毒PreS1肽体外免疫.分别从免疫和未经免疫的淋巴细胞提取RNA,扩增抗体基因,构建大容量天然单链抗体(scFv)噬菌体展示文库和体外免疫scFv抗体库.以PreS1肽进行3轮淘选后,抗原抗体反应结果显示,从免疫库中获得了亲和力10-7～10-8 M的抗乙型肝炎病毒PreS1的单链抗体,高于天然库的结果(10-6～10-7 M).测序结果表明两株抗体均为人抗体.为基因工程抗体用于临床治疗乙型肝炎奠定基础.同时证明淋巴细胞体外免疫方法构建的免疫抗体库优于大容量天然抗体库.     

黄酮类化合物协同增效作用研究进展

黄酮类化合物是自然界中较为常见的植物化学素，具有多种生理活性与功能，已经被广泛应用于食品、医药等诸多领域中。但是天然黄酮类化合物发挥作用有限，单独作用时功能效果不显著，因此，对目前来说增强黄酮类化合物功效的发挥是迫切需要解决的问题。本文综述了天然产物黄酮类化合物在与自然界其他天然分子间协同增效方面的研究进展，对协同作用的评价方法进行了归纳，并对协同效应相关机制进行了总结，以期为以黄酮类化合物为主的天然功能食品与药品的复配开发提供参考。     

天然化合物抗真菌活性研究进展

近年来,真菌感染患者的发病率和死亡率持续上升,但现有抗真菌药物种类依然非常少,并且耐药现象的出现使临床可选择的抗真菌药物变得更加有限.因此,对新的抗真菌药物的开发迫在眉睫,从天然产物中寻找新型高效的抗真菌药物成为目前的研究热点之一.从天然产物中筛选出具有抗真菌活性的天然化合物,有助于扩大治疗真菌感染疾病的可选药物种类,减少耐药的发生.该文归纳现有报道的具有抗真菌活性的化合物,根据其不同来源及不同化学结构进行分类,阐明不同类别天然化合物的抗真菌作用机制,为开发新型高效抗真菌药物提供前体结构及抗真菌新靶点.     

激活低氧诱导因子的天然小分子化合物研究进展

缺血及随之发生的低氧损伤严重危害人类健康.大量研究显示,低氧诱导因子(hypoxia inducible factor-1,HIF-1)的激活通过调控促存活基因,改善缺血/低氧损伤的预后.近年来,针对具有激活HIF-1的天然小分子化合物的研究受到了越来越多的关注.该类化合物具有毒性低、副作用少等优点,具有发展成为防治缺血/低氧药物的潜力.本文简单介绍了HIF-1的调控机制,并总结了近年来发现的具有激活HIF-1作用的天然小分子化合物及其作用机制.     

Bisphenol-A Can Inhibit the Enzymatic Activity of Human Superoxide Dismutase

Bisphenol-A (BPA), a synthetic xenoestrogen, is currently being used to produce a wide variety of consumer products. Humans as well as animals are exposed to this ubiquitous compound through ingestion, inhalation, and dermal exposure. The effect of this compound on superoxide dismutase (SOD), an antioxidant enzyme, isolated from human blood was studied using an enzyme inhibition assay. The mode of interaction of BPA on SOD was investigated using modeling and docking studies. Purified human SOD from erythrocytes was used to study the enzyme inhibition assay of BPA. Molecular level interactions of BPA on SOD were also analyzed by modeling and docking studies. Our study demonstrates that BPA has an inhibitory effect on SOD. The docking results showed that it could bind to the active site residues of SOD and could interfere with the catalytic activity of the enzyme. Our study reveals for the first time that BPA can directly inhibit the enzymatic activity of human SOD and thus impairs the free radical scavenging mechanism.     

Kaempferol: a new immunosuppressant of calcineurin

Calcineurin (CN), the Ca(2+)/calmodulin (CaM)-dependant protein phosphatase, is the target for immunosuppressive drugs cyclosporine A (CsA) and FK506. These immunosuppressants can inhibit CN activity after binding with respective immunophilins. Based on the model of screening by using p-nitrophenyl phosphate as a substrate for preliminary screening and (32)P-labeled 19-residue phosphopeptide as a specific substrate for final determination, we found Kaempferol, a natural flavonol, could inhibit CN activity in purified enzyme and Jurkat T-cells. Unlike CsA and FK506, CN inhibition by kaempferol is independent of matchmaker protein and the inhibitory manner is noncompetitive. Through investigation of inhibitions for CNA and a series of its truncated mutants, we suggested that Kaempferol could directly act on the catalytic domain. Data also indicated that the CN inhibition by kaempferol could be enhanced when the enzyme was activated in the presence of CaM and CNB. CNB is necessary for mediating inhibition of enzyme by kaempferol. The result of RT-PCR also indicated that kaempferol had an inhibitory activity against IL-2 gene expression in activated Jurkat cells. All data suggested that kaempferol could be a new immunosuppressant of CN.     

Synthesis, molecular docking and biological evaluation of 1,3,4-oxadiazole derivatives as potential immunosuppressive agents

A series of novel 1,3,4-oxadiazole derivatives (5a-5s) have been designed, synthesized and evaluated for their immunosuppressive activity. Most of these synthesized compounds were proved to have potent immunosuppressive activity and low toxicity. Among them, compounds (5m-5r) showed the most potent biological activity against lymph node cells. The results of flow cytometry (FCM) and western blotting demonstrated that compound 5q induce cell apoptosis by the inhibition of PI3K/AKT pathway. Molecular docking was performed to position compound 5q into PI3Kγ binding site in order to explore the potential target.     

Molecular interaction between 4-aminoantipyrine and catalase reveals a potentially toxic mechanism of the drug

4-Aminoantipyrine (AAP) is scarcely administered as an analgesic drug because of the potential side effects. The residue of AAP in the environment possesses a potential threat to human health. In this article, the binding mode of AAP with the important antioxidant enzyme catalase (CAT) was investigated using spectroscopic and molecular docking methods. AAP can interact with CAT to form an AAP-CAT complex. The binding constant, number of binding sites and thermodynamic parameters were measured, which indicated that AAP could spontaneously bind with CAT through electrostatic forces with one binding site. Molecular docking results revealed that AAP bound into the CAT central cavity. UV-visible absorption, synchronous fluorescence and circular dichroism (CD) results provide data concerning conformational and some microenvironmental changes of CAT. Furthermore, the binding of AAP can inhibit CAT activity in erythrocytes. The present study provides direct evidence at a molecular level to show that exposure to AAP could induce changes in the enzyme CAT structure and function. The estimated methods in this work can be applied to characterize interactions of enzyme systems and other pollutants and drugs.     

Fragment splicing-based design,synthesis and safener activity of novel substituted phenyl oxazole derivatives

Fragment splicing is a primary strategy in the design and optimization of leading compound toward new skeleton with target bioactivity. Herein a series of novel substituted phenyl oxazole derivatives were designed via fragment analysis and coupling strategy that led to highly potent and bio-selective herbicide safener. The biological tests showed that most of the compounds could enhance the maize growth index, glutathione content and anti-reverse enzyme glutathione S-transferase activity in vivo. The molecular docking model exhibited that the novel compound could compete with chlorsulfuron binding to the herbicide target enzyme, which consequently attained the herbicide detoxification. Especially compound I-f displayed the best activities than commercial safener isoxadifen-ethyl and other compounds. The present work demonstrates that the synthesized compounds could be developed as potential candidates for the discovery of novel herbicide safeners in the future.     

The interprotein scoring noises in glide docking scores

Small molecule drugs are rarely selective enough to interact solely with their designated targets. Unintended "off-target" interactions often lead to side effects, but also serendipitously lead to new therapeutic uses. Identification of the off-targets of a compound is therefore of significant value to the evaluation of its developmental potential. In computational biology, the strategy of "reverse docking" has been introduced to predict the targets of a compound, which uses a compound to virtually screen a library of proteins, reversing the bait and prey in "normal" docking screenings. The present study shows that, in reverse docking, additional optimization of the scoring function may help to improve the target prediction accuracy. In a case study with the Glide scores, we found that only 57% of the ligand-protein relationships could be correctly identified in a library of 58 complexes whose crystal binding conformations were all able to be accurately reproduced. This was likely a result of the constant over- or under-estimation of the scores for specific proteins. In other words, there were interprotein noises in the Glide scores. Introducing a correction term based on protein characteristics improved the target-prediction accuracy by 27% (57-72%). It is our hope that this focused discussion on the Glide scores would invite further efforts to characterize and normalize this type of interprotein noises in all docking scores, so that better target prediction accuracy can be achieved with the strategy of reverse docking.     

Isogarcinol Is a New Immunosuppressant

Calcineurin (CN), a unique protein phosphatase, plays an important role in immune regulation. In this study we used CN as a target enzyme to investigate the immunosuppressive properties of a series of natural compounds from Garcinia mangostana L., and discovered an active compound, isogarcinol. Enzymatic assays showed that isogarcinol inhibited CN in a dose-dependent manner. At concentrations resulting in relatively low cytotoxicity isogarcinol significantly inhibited proliferation of murine spleen T-lymphocytes induced by concanavalin A (ConA) and the mixed lymphocyte reaction (MLR). In addition, it performed much better in acute toxicity tests and via oral administration in mice than cyclosporin A (CsA), with few adverse reactions and low toxicity in experimental animals. Oral administration of isogarcinol in mice resulted in a dose-dependent decrease in delayed type hypersensitivity (DTH) and prolonged graft survival in allogeneic skin transplantation. These findings suggest that isogarcinol could serve as a new oral immunomodulatory drug for preventing transplant rejection, and for long-term medication in autoimmune diseases.     

Effect of different immunosuppressive drugs on calcineurin and its mutants

Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis.Their phosphatase activity and the corresponding solution conformation were examined.Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immunosuppressive drugs with CN.The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN.Furthermore,circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein.Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN,and had no effects on the phosphatase activity of mutants in Loop7 region,which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN.Examination of the activities of these mutants resulted in the presence of immunosuppressive component from traditional Chinese drugs.The component of Chinese drug,ZIP1,could directly inhibit both CN and CN mutants without drug binding protein.These results suggest that the Loop7 region is an important structural area involved in the inhibition by CyP-CsA.It is valuable to further study the inhibition by ZIP1.     

Homology modeling,molecular docking and spectra assay studies of sterol 14α-demethylase from <Emphasis Type="Italic">Penicillium digitatum</Emphasis>

Sterol 14α-demethylase from Penicillium digitatum (PdCYP51) is a prime target of antifungal drugs for citrus disease in plants. To design novel antifungal compounds, a homology model of PdCYP51 was constructed using the recently reported crystal structure of human CYP51 as the template. Molecular docking was performed to investigate the interaction of four commercial fungicides with the modeled enzyme. The side chain of these compounds interplayed with PdCYP51 mainly through hydrophobic and van der Waals interactions. Biochemical spectra analysis of inhibitors combined with PdCYP51 are also compatible with the docking results. This is the first molecular modeling for PdCYP51 based on the eukaryotic crystal structure of CYP51. The structural information and binding site mapping of PdCYP51 for different inhibitors obtained from this study could aid in screening and designing new antifungal compounds targeting this enzyme.