螃蟹甲内生真菌Chaetosphaeronema sp.的化学成分研究

目的 研究一株来源于藏药螃蟹甲内生真菌 PHY-24的化学成分。 方法 采用麦麸培养基,对内生真菌 PHY-24进行放大培养,通过硅胶柱色谱、MCI 柱色谱、ODS 柱色谱、Sephadex LH-20柱色谱、高效液相色谱等对其发酵液中的化学成分进行分离纯化,利用 NMR、MS 等波谱方法进行化学成分的结构鉴定,并测定这些成分抗 HIV-1整合酶链转移反应活性。 结果 从内生真菌 PHY-24发酵液的乙酸乙酯提取部分分离并鉴定了4个化合物,分别是：integrastatin B（1）、2-乙酰基-3,5-二羟基-苯乙酸（2）、curvulin （3）、O-methylcurvulinic acid（4）。其中化合物（1）和（2）具有抗 HIV-1整合酶链转移反应活性,其 IC50分别为6.22和75.1μmol/L。 结论 4个化合物均为从此属真菌中首次分得,其中化合物（1）、（2）具有抗 HIV-1整合酶链转移反应活性。化合物（2）的活性为首次报道。     

卡斯帕酶-3抑制剂的高通量筛选及活性化合物F03ZA-575的初步研究

目的筛选并初步研究微生物来源的卡斯帕酶-3(Caspases-3)抑制剂活性化合物。方法使用E.coli异源表达的卡斯帕酶-3作为靶酶,建立基于Caspase-3酶抑制剂的体外高通量筛选模型并对微生物来源的共计10026个次级代谢产物提取物进行了筛选。使用有机溶剂萃取、硅胶柱和LH-20柱层析、高压液相分离等方法从代谢产物中分离活性化合物并经各种理化性质及NMR分析等对活性化合物的结构进行确定。结果从10026个微生物来源的代谢产物中筛选获得了10个阳性样品。其中,从1株真菌的代谢产物中分离得到的化合物F03ZA-575对Caspase-3酶显示出较强的抑制活性,结构解析确认该化合物与Duclauxin同质。结论该化合物对Caspase-3酶的抑制活性将有助于揭示其抗肿瘤作用的机制。     

双二酮酸结构HIV-1整合酶抑制剂的合成及活性研究

目的设计合成双二酮酸类化合物,并探讨此类化合物抑制HIV-1整合酶活性的构效关系。方法以甲基取代苯乙酮为起始原料,经溴代后与羟基取代苯乙酮反应生成乙酰基苄氧苯乙酮,然后在氢化钠作用下,脱去α氢后进攻草酸二乙酯生成双二酮酸酯,水解脱去酯基生成双二酮酸;经1H-NMR、IR和MS验证化合物结构。ELISA法测定目标化合物对HIV-1整合酶的抑制活性;采用高通量荧光法测定目标化合物对HIV-1整合酶3'加工过程的抑制活性;测定目标化合物对HIV假病毒的细胞活性。结果合成了6个全新结构的二酮酸类化合物;体外整合酶活性评价结果表明,6个目标化合物对HIV-1整合酶的活性IC50≤20μg/ml,双二酮酸之间距离稍短的5a、5c、5d对3'加工过程有一定的活性,IC50约为40μg/ml。结论 6个新化合物对HIV-1的活性主要表现在链转移过程。     

HIV-1整合酶酶联免疫吸附试验的建立和应用

目的：建立一种检测人类免疫缺陷病毒1型(HIV-1)整合酶的酶联免疫吸附试验(ELISA)方法,用于筛选HIV-1整合酶抑制剂。方法：将质粒F185K／C2280SIN1—288转化到大肠杆菌中,经IPTG诱导表达,柱亲和层析纯化,获得HIV-1整合酶融合蛋白。建立酶联免疫吸附试验(ELISA)方法。与^3H同位素标记方法比较,     

木霉属真菌拮抗金钗石斛病原菌的研究

目的考察石斛属药用植物内生真菌拮抗软腐病的真菌。方法将179株内生真菌进行金钗石斛软腐病生物防治盆栽试验,以获得拮抗病原菌终极腐霉的内生真菌。将具有拮抗效果的真菌在PDA培养基中与终极腐霉进行双重培养平皿对峙试验并对其显微形态进行观察,利用3,5-二硝基水杨酸法检测真菌的β-1,3和β-1,4葡聚糖酶活性。结果 T.koningiopsis与T.rogersonii在盆栽试验和双重培养平皿对峙试验中对终极腐霉具有抑制作用。T.koningiopsis与T.rogersonii菌丝将终极腐霉菌丝包围并将其破坏;两者β-1,3和β-1,4葡聚糖酶活性在所测试的5种真菌中最高。结论 T.koningiopsis与T.rogersonii可有效抑制终极腐霉;由两者分泌的β-1,3和β-1,4葡聚糖酶可能在破坏终极腐霉细胞壁方面发挥作用。T.koningiopsis和T.rogersonii可作为潜在的生防菌发挥拮抗终极腐霉作用。     

以HIV-1 IN-LEDGF/p75相互作用为靶点的抑制剂筛选

目的晶状体上皮源性生长因子p~(75)蛋白(LEDGF/p75)与HIV-1整合酶(IN)之间的蛋白-蛋白相互作用是开发抗HIV-1药物的有效靶点,本文旨在寻找以HIV-1IN-LEDGF/p75相互作用为靶点的小分子抑制剂。方法采用均相时间分辨荧光技术(HTRF)筛选了799个化合物,比对IN-LEDGF/p75相互作用的抑制活性。结果发现5个化合物——肾上腺酮、6-溴-1,2-二氢萘-1,2-二酮、头孢噻吩、根皮含柘树呫吨酮L和迷迭香酸对该相互作用表现出不同程度的抑制作用,其IC50值分别为12.3、22.7、26.2、18.5和1.13μmol/L。结论为HIV-1IN-LEDGF/p75相互作用抑制剂的发现以及新的抗HIV-1药物的开发提供了重要基础。     

抗艾滋病药物设计的新战略:逆转录病毒整合酶抑制因子

近年来,随着HIV病毒分子生物学研究的不断深入以及多种治疗途径的综合运用,已使艾滋病人的预后得到了明显的改善.虽然目前的治疗可使病人HIV的滴度明显降低,但HIV仍然存在于病人的外周血单核细胞、静止T淋巴细胞等细胞中.针对HIV的逆转录酶和蛋白酶抑制因子已设计、筛选了多种药物并投入临床应用,但同时也导致抗药性病毒的产生,由于存在上述这些问题,有必要寻找新的病毒分子组份以便针对其设计、筛选新的特异、高效的抗HIV药物.HIV-1整合梅(HIV-1 intergrase)即是理想的该类靶分子.本文就HIV整合酶抑制因子类药物研究的最新进展作一综述.     

第二代HIV-1整合酶抑制剂筛选靶点概述

<正>HIV-1整合酶是病毒复制所必需的酶[1]。在临床上,使用HIV-1整合酶(integrase,IN)抑制剂来治疗HIV感染被证明是非常有益的。目前,FDA批准上市的第一个HIV-1整合酶抑制剂raltegravir已作为一线药物用于临床[2-3]。与raltegravir作用机制相同的另外一个药物elvitegravir目前正处于III期临床试验阶段[4]。Raltegravir和elvitegravir作为第一代整合酶抑制剂,其作用机制是抑制HIV-1整合酶链转移反应[5-6],因此,它们被称为链转移反应抑制剂     

以蛋白激酶A为靶点的抗结核药物高通量筛选模型的建立和应用

目的 建立以蛋白激酶 A 为靶点的抗结核药物高通量筛选模型,应用该模型筛选具有特异性酶活抑制活性的微生物发酵液粗提物样品。 方法 以结核分枝杆菌 H37Rv 基因组 DNA 为模板,扩增目的基因片段 pknA,构建表达载体 pET43.1a-pknA,在大肠杆菌中克隆表达了重组 MTB PknA 蛋白;采用三步级联的反应方法,利用还原型烟酰胺腺嘌呤二核苷酸到氧化型烟酰胺腺嘌呤二核苷酸这一反应最大吸光值波长的变化,建立和优化蛋白激酶 A 抑制剂高通量药物筛选模型。 结果 成功构建了表达载体 pET43.1a-pknA;建立了稳定灵敏,可用于靶向结核分枝杆菌蛋白激酶 A 的抗结核药物高通量筛选模型;利用该模型对4000个微生物发酵液粗提物样品进行筛选,最终得到21个抑制蛋白激酶 A 活性的阳性样品,阳性率0.53%;以耻垢分枝杆菌和海分枝杆菌为检定菌,平板纸片法检测阳性样品的抗分枝杆菌活性,然后对阳性样品的细胞毒性和酶活抑制特异性进行评价后,最终得到8个阳性样品,其中 I10AA-02916、I09AA-02717、I09AB-02729、I08AB-00801这4个阳性样品酶活抑制特异性、抗菌活性均较好,且细胞毒性较低。 结论 建立了高稳定性的以蛋白激酶 A 为靶点的抗结核药物高通量筛选模型,应用该模型所得到的发酵液阳性样品值得进一步研究。     

介绍HIV-1整合酶抑制剂研究中几种检测方法

抗HIV-1整合酶（Integrase,IN）抑制剂的研发主要依赖于体外筛选方法,因此设计可靠稳定的筛选方法是新药研究和开发的基础.     

HIV-1整合酶酶联免疫吸附试验及其抑制剂的研究

目的　建立一种检测人类免疫缺陷病毒 1型 (HIV - 1 )整合酶的酶联免疫吸附试验(ELISA)方法 ,用于筛选和研究HIV - 1整合酶抑制剂。方法　将质粒F1 85K C2 80SIN1 2 88转化到大肠埃希菌中 ,经IPTG诱导表达 ,柱亲和层析纯化 ,获得HIV 1整合酶融合蛋白。建立酶联免疫吸附试验方法测定其生物学活性 ,与3 2 P同位素标记方法比较 ,并用ELISA方法筛选HIV 1整合酶的抑制剂。结果　SDS PAGE电泳分析显示 ,相对分子质量 30 0 0 0上方有HIV 1整合酶融合蛋白条带出现。ELISA及3 2 P同位素标记法证实 ,此融合蛋白对于特异底物具有 3′切割和链转移活性。ELISA反应的平均P N值为 2 836± 0 1 61 ,批内及批间变异系数 (CV)分别为 4 63 %和 5 89%。检测到中药丹参提取物CEH等有抑制整合酶的活性 ,CEH的大孔树脂洗脱物CEHL活性提高。结论　ELISA法检测HIV 1整合酶活性技术简单 ,快速 ,重复性好 ,无同位素污染 ,可用于HIV 1整合酶为靶点的抑制剂的筛选及抗 HIV药物作用机理的研究。     

Control of HIV through the inhibition of HIV-1 integrase: a medicinal chemistry perspective

This article reviews the current status of classes of HIV-1 integrase enzyme inhibitors. These classes include peptide-based inhibitors, natural products, polyhydroxylated aromatics, diketo acids, naphthyridines, and sulfonated compounds including sulfonic acids. Discussions of structure activity relationships are presented and include the current overview of the structure-based model, suitable for the further design and development. To date, the advances in the medicinal chemistry of HIV-1 integrase inhibitors have relied mostly on ligand-based designs leading to most displaying similar binding interactions within the active site or at the dimer interface. This paves the way for single enzyme mutations rendering entire compound classes inactive and thus, the requirement for second and third generation inhibitors with novel modes of binding is apparent. To facilitate future structure-based drug design efforts, a model of the biologically relevant structure of the HIV-1 integrase enzyme, a dimer of dimers has also been discussed.     

In vitro studies on antifungal activity of tea (Camellia sinensis) and coffee (Coffea arabica) against wood-rotting fungi

The present study reports the sensitivity of ten different wood-rotting fungi towards eight samples of tea and two samples of coffee. The tea samples included five CTC black teas, one orthodox black tea and two green teas. The different fungi tested were sensitive to different tea and coffee samples to variable levels. Green tea caused the maximum growth inhibition and it was 100% in case of Phanerochaete chrysosporium and Sporotrichum pulverulentum. Of the black tea samples, orthodox black tea was more effective inhibitor for all the fungi tested. Phlebia radiata was the most sensitive fungus to all the black tea samples except Lipton Cheers. The growth of Daldinia concentrica was least affected by all the black tea samples. The two coffee samples showed better inhibition than CTC black tea samples. The inhibitory effect of tea and coffee extracts was greatly reduced on their filtration. All the tea and coffee samples tested showed more than 50% reduction in their inhibitory activity. The inhibitory effect was completely lost in spent black tea leaves. The antifungal effect of pure caffeine was tested at different concentrations. Majority of the fungi were totally inhibited at a concentration of 0.3% whereas, D. concentrica, P. radiata and P. palustris showed complete inhibition at a concentration of 0.5 percent.     

A genotypic assay for the amplification and sequencing of integrase from diverse HIV-1 group M subtypes

Recently, the Food and Drug Administration (FDA) of the USA approved the first integrase inhibitor for inclusion in treatment regimens of HIV-1 patients failing their current regimens with multi-drug resistant strains. However, treatment failure has been observed during integrase inhibitor-containing therapy. Several mutational pathways have been described with signature mutations at integrase positions 66, 92, 148 and 155. Therefore, a genotypic assay for the amplification and sequencing of HIV-1 integrase was developed. The assay displayed a detection limit of 10 HIV-1 III(B) RNA copies/ml plasma. As the HIV-1 pandemic is characterised by a large genetic diversity, the new assay was evaluated on a panel of 74 genetically divergent samples belonging to the following genetic forms A, B, C, D, F, G, J, CRF01-AE, CRF02-AG, CRFF03-AB, CRF12-BF and CRF13-cpx. Their viral load ranged from 178 until >500,000 RNA copies/ml. The amplification and sequencing was successful for 70 samples (a success rate of 95%). The four failures were most probably due to low viral load or poor quality of RNA and not to subtype issues. Some of the sequences obtained from integrase inhibitor-na?ve patients displayed polymorphisms at integrase positions associated with resistance: 74IV, 138D, 151I, 157Q and 163AE. The relevance of these polymorphisms in the absence of the signature mutations remains unclear.     

Quantification of HIV-1 group M (subtypes A-G) and group O by the LCx HIV RNA quantitative assay

Human immunodeficiency virus type 1 (HIV-1) genetic diversity presents a challenge to nucleic acid-based assays with regard to sensitivity of detection and accuracy of quantification. The Abbott LCx HIV RNA Quantitative assay (LCx(R) HIV assay), a competitive RT-PCR targeting the pol integrase region, was evaluated using a panel of 297 HIV-1 seropositive plasma samples from Cameroon, Uganda, Brazil, Thailand, Spain, Argentina and South Africa. The panel included group M subtypes A-G, mosaics, and group O based on sequence analysis of gag p24, pol integrase, and env gp41. The LCx HIV assay quantified 290 (97.6%) of the samples, including all the group O samples tested. In comparison, the Roche AMPLICOR HIV-1 MONITOR test versions 1.0 and 1.5 quantified 67.3 and 94.6% of the samples, respectively. No group O specimens were quantified by either version of AMPLICOR HIV-1 MONITOR. Seven specimens were below the detectable limits of all the three assays. The LCx HIV assay had fewer nucleotide mismatches at primer/probe binding sites as compared with both AMPLICOR HIV-1 MONITOR tests. The high degree of nucleotide conservation within the pol target region enables the LCx HIV assay to efficiently quantify the HIV-1 subtypes A-G and the most genetically diverse HIV-1, group O.