以肽脱甲酰基酶为靶点的抗结核药物高通量筛选模型的建立和应用

目的建立以肽脱甲酰基酶(PDF)为靶点的抗结核药物高通量筛选模型,应用该模型筛选得到活性微生物发酵液粗提物样品。方法以结核分枝杆菌H37Rv基因组为模板,扩增肽脱甲酰基酶的基因片段def,构建表达载体pET-28a-def,表达并纯化结核分枝杆菌PDF酶;基于PDF水解三肽底物for-Met-Ala-Ser释放出游离NH2,而游离NH2可与荧光胺反应产生荧光的原理,利用测定所产生荧光值的方法,建立高通量药物筛选模型;使用该模型对12400个微生物发酵液粗提物样品进行筛选,同时以耻垢分枝杆菌为检定菌,平板纸片法检测样品的抗菌活性,并检测所得阳性样品的细胞毒性。结果成功构建了表达载体pET-28a-def;所建立的模型稳定可行,可用于以肽脱甲酰基酶为靶点的抗结核药物的高通量筛选;用该模型对12400个微生物发酵液粗提物样品进行筛选,最终得到8个对肽脱甲酰基酶抑制活性和抗耻垢分枝杆菌活性均较好的阳性样品,阳性率0.06%;其中5个样品的细胞毒性较小。结论建立了灵敏度好、稳定性高的结核分枝杆菌肽脱甲酰基酶抑制剂高通量药物筛选模型,应用该模型所得到的阳性样品具有进一步深入研究的意义。     

以丙氨酸消旋酶为靶点的高通量抗结核药物筛选模型的建立及应用

目的建立以结核分枝杆菌丙氨酸消旋酶为靶点的新型高通量抗结核药物筛选模型,筛选丙氨酸消旋酶的抑制剂,获得以丙氨酸消旋酶为靶点的新型抗结核药物先导物。方法以结核分枝杆菌H37Rv基因组为模板,pET28a表达质粒为载体,将alr基因克隆至pET28a,构建pET28a::alr重组表达质粒,表达并纯化得到重组结核分枝杆菌丙氨酸消旋酶;通过测定反应产物NADH在340 nm处光密度变化速率,检测酶反应活性,构建并优化该酶抑制剂的高通量筛选模型;应用该模型对化合物库进行筛选;测定活性化合物IC50以及对结核分枝杆菌的MIC。结果成功构建了结核分枝杆菌alr基因的表达载体;得到了纯度较高的重组丙氨酸消旋酶,测得该酶的比活力为13.53 kU/mg;所建立的丙氨酸消旋酶高通量筛选模型稳定性高,符合高通量筛选的要求;通过对70 000个化合物进行筛选,得到了5个活性较高的化合物,其中,IMB-XZ5对结核分枝杆菌的MIC为4~8μg/ml,且对结核分枝杆菌的作用具有较高的特异性。结论建立了稳定性好、灵敏度较高的结核分枝杆菌丙氨酸消旋酶抑制剂高通量筛选模型,应用该模型筛选得到了具有较好抗结核活性的丙氨酸消旋酶抑制剂。     

以金色分枝杆菌为模式菌株筛选抗结核活性化合物可行性研究

目的评估以金色分枝杆菌作为结核分枝杆菌模式菌株筛选抗结核抑制剂的可行性。方法以结核分枝杆菌标准菌株H37Rv建立细胞水平的抑制剂高通量筛选模型,对本单位化合物库部分样品进行筛选,获得具有抗结核活性的化合物;进一步比较模式菌株金色分枝杆菌、耻垢分枝杆菌、海分枝杆菌及谷氨酸棒状杆菌对具有较强抗结核活性样品的敏感性差异。结果利用基于结核分枝杆菌建立的全细胞筛选模型,从本单位化合物库的5万个样品中筛选得到67个最低抑菌浓度≤5μg/ml的化合物。对金色分枝杆菌、耻垢分枝杆菌、海分枝杆菌和谷氨酸棒状杆菌有抑制活性的样品分别有22个(32.84%)、10个(14.93%)、12个(17.91%)和6个(8.96%),其中抑菌活性与抗结核活性差异在4倍以内的样品分别有16个(72.73%)、5个(50%)、7个(58.33%)和3个(50%)。结论相对于其他3种模式菌株,金色分枝杆菌对本单位样品库化合物的敏感性与结核分枝杆菌的最为接近,以金色分枝杆菌为模式菌株开展抑制剂筛选研究更有可能获得具有抗结核活性的化合物。     

以结核分枝杆菌H_(37)Rv莽草酸脱氢酶为靶点的高通量筛选模型的建立及应用

目的建立以结核分枝杆菌莽草酸脱氢酶为靶点的新型抗结核药物高通量筛选模型;用此模型筛选莽草酸脱氢酶抑制剂;进一步评价化合物对莽草酸脱氢酶活性的影响。方法表达并纯化结核分枝杆菌H37Rv莽草酸脱氢酶;利用还原型辅酶II(NADPH)在溶液中的光吸收,测定酶的活性,构建了该酶抑制剂的高通量筛选模型;用Z′因子法评价该模型的可靠性,并对5万余个化合物进行筛选;测定了各抑制剂的IC50并对抑制剂6186050的酶抑制动力学进行了研究;用菌液稀释法评价了抑制剂对某些临床分离菌株包括耐药菌株的影响。结果得到了重组莽草酸脱氢酶;测得比活力为20987U/mg,所建的莽草酸脱氢酶高通量筛选模型Z′因子为0.76,符合高通量筛选的要求;对5万余个化合物进行筛选得到9个抑制率较高的化合物;抑制剂6186050为竞争性可逆抑制剂;抑制剂6230384和6186050对海分枝杆菌的最低抑菌浓度(MIC)都是32μg/ml。结论建立了稳定性好、灵敏度较高的结核分枝杆菌莽草酸脱氢酶抑制剂高通量药物筛选模型,应用该模型筛选得到的抑制剂可能具有抑菌活性。     

埃博拉病毒进入抑制剂筛选模型的建立与应用

目的旨在以埃博拉病毒(EBOV)包膜糖蛋白(GP)为靶点,建立埃博拉病毒进入抑制剂高通量筛选模型,应用该模型筛选具有特异性抑制埃博拉病毒进入的活性样品。方法利用细胞水平重组病毒技术,分别用两株不同爆发时间的Zaire-EBOV的GP与HIV核心质粒(p NL4-3.Luc)共表达,制备重组病毒EBOV-GP-Old/HIV-luc和EBOV-GP-New/HIV-luc。利用ELISA和荧光素酶检测技术,优化病毒产量、感染性、感染剂量及荧光活性测定时间等因素,建立药物筛选模型。利用统计学参数及阳性化合物评估该模型的稳定性和灵敏性。应用该模型,对242个样品进行初步筛选,并通过与水泡性口膜炎病毒包膜蛋白包装的重组病毒VSVG/HIV-luc进行比较,以判断样品的特异性。结果该模型可用于靶向GP蛋白的埃博拉病毒进入抑制剂的高通量筛选,并获得4种具有特异性抗埃博拉进入活性的样品。结论该模型可用于抗EBOV进入药物的高通量筛选,应用该模型获得的阳性样品有助于抗EBOV药物的发现。     

抗寨卡病毒药物筛选模型的建立及其应用

目的建立寨卡病毒抑制剂高通量筛选模型,为筛选和评价抗寨卡病毒化合物提供技术手段。方法利用CCK-8法测定寨卡病毒感染后细胞活性,间接反应胞内病毒复制水平。进而对分析方法进行优化,建立可定量病毒复制能力的药物筛选模型,并利用统计学分析对模型的稳定性和灵敏性进行评价。应用该模型对32 000个微生物发酵液进行初步筛选,验证该方法的可行性。结果成功建立寨卡病毒抑制剂高通量筛选模型,该筛选模型具有较好的稳定性和灵敏度,Z'因子为0.733,CV为5.52%,S/B为20.21,S/N为14.18。利用该模型筛选得到阳性样品102个,对其中I07A-01164进行抗病毒活性验证,测定其稀释4倍后可以抑制50%以上的病毒复制。结论建立了抗寨卡病毒化合物的高通量筛选模型,可用于寨卡病毒抑制剂的筛选和活性初步评价,有助于抗寨卡病毒药物的发现。     

核糖体蛋白L12-L10相互作用抑制剂的筛选及其抗结核活性的研究

目的以L12-L10相互作用为靶点筛选具有抗结核活性的先导化合物。方法应用酵母双杂交模型AH109(pAD-L12+pBD-L10)通过生长抑制方法筛选阳性化合物,以AH109(pAD-T+pBD-53)作为对照;通过96孔板法检测阳性化合物对耻垢分枝杆菌的抑制活性;应用定量微孔板快速显色法(MABA)检测抗结核杆菌活性;通过β-半乳糖苷酶活性定量检测判断阳性化合物在模型上对L12-L10相互作用的阻断活性;应用体外蛋白表达系统检测阳性化合物对蛋白表达的抑制作用;应用平皿二倍稀释法检测阳性化合物的药敏作用。结果筛选到4个在模型上具有活性的阳性化合物,其对耻垢分枝杆菌具有比较好的抑制活性,其最小抑制浓度(MIC)在3.125~12.5μg/ml之间;其中IBM-T275对结核分枝杆菌标准株和临床分离株均具有比较好的抑制活性,MIC在5~10μg/ml之间,而对细菌抑制活性较低,其MIC均在64μg/ml以上;IBM-T275能够抑制酵母模型内β-半乳糖苷酶的表达,并且能够体外抑制蛋白表达,其IC50为12.57μg/ml。结论筛选到1个具有抗结核杆菌活性的阳性化合物,其抗结核活性可能与阻断L12-L10蛋白相互作用相关。     

结核分枝杆菌FtsZ抑制剂的筛选和活性研究

目的筛选结核分枝杆菌FtsZ的特异性抑制剂,为抗结核药物的研发提供先导化合物。方法应用本实验室已经建立的结核分枝杆菌FtsZ抑制剂筛选模型对化合物库进行筛选,获得能够抑制FtsZ的化合物202E,对其进行IC50以及分子水平活性测定,并利用DS 4.0软件将化合物202E与FtsZ的活性位点进行对接。结果化合物202E能够抑制结核分枝杆菌FtsZ的GTP酶活性,其IC50为15.46μmol/L。202E还能够抑制FtsZ蛋白的聚合。通过分子对接,发现202E能够与FtsZ的GTP结合位点结合,抑制FtsZ的GTP酶活性。结论化合物202E是活性较好的结核分枝杆菌FtsZ抑制剂。     

抗耐药性结核病新药的先导化合物筛选

目的 采用结核分枝杆菌(Mtb)H37Rv对从上海陶塑生物科技有限公司购买的生物活性化合物库L4000进行抑制剂筛选,以获得具有成药前景的抗结核先导化合物.方法 采用已经建立的Mtb H37Rv细胞水平的抑制剂高通量筛选模型对化合物库L4000的7285个样品进行筛选,并测定初筛阳性化合物对H37Rv以及临床多药耐药结...     

以次黄嘌呤单核苷酸脱氢酶为靶点的新型抗结核药物高通量筛选模型的建立及应用

目的 建立以结核分枝杆菌次黄嘌呤单核苷酸脱氢酶为靶点的新型抗结核药物高通量筛选模型.方法 以结核分枝杆菌H37Rv基因组为模板,pBEV表达质粒为载体,将guaB2基因克隆至pBEV以构建pBEV::guaB2重组表达质粒,表达并纯化重组的结核分枝杆菌次黄嘌呤单核苷酸脱氢酶;建立以测定反应体系340 nm吸光值变化速率...     

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。     

Specific identification of Mycobacterium tuberculosis with the luciferase reporter mycobacteriophage: use of p-nitro-alpha-acetylamino-beta-hydroxy propiophenone.

We have previously described a luciferase reporter mycobacteriophage (LRP) assay that can detect Mycobacterium tuberculosis and characterize mycobacterial drug susceptibility patterns within 24 to 48 h in positive cultures. One drawback of this LRP protocol is the ability of the recombinant mycobacteriophage phAE40 to infect a variety of Mycobacterium species, thus limiting its specificity for the detection of M. tuberculosis. In this study, we have (i) explored the host range of phAE40, (ii) developed a modified LRP assay that exploits the selective inhibitory effect of the compound p-nitro-alpha-acetylamino-beta-hydroxy propiophenone (NAP) against members of the M. tuberculosis complex to differentiate between the tubercle bacillus and other mycobacterial species, and (iii) tested over 300 samples, including primary clinical isolates and drug-resistant strains of M. tuberculosis, demonstrating the ability of the NAP-modified LRP assay to identify M. tuberculosis complex organisms with high degrees of sensitivity and specificity.     

噬菌体生物扩增法测定痰标本结核分枝杆菌利福平耐药性

目的建立快速测定痰标本中结核分枝杆菌利福平（RFP）耐药性的噬菌体生物扩增法，探讨其在临床应用的可行性。方法噬菌体生物扩增法测定362份涂阳痰标本结核分枝杆菌对RFP的耐药性，并与罗氏绝对浓度法结果进行比较，对不符合的样本，用基因芯片的方法分析rpoB基因的突变情况。结果362份涂阳痰标本，培养阳性360份，菌种初步鉴定有17份样本为非结核分枝杆菌，噬菌体生物扩增法RFP耐药为123份，敏感的196份，两法结果一致的为88．6％，如以绝对浓度法药敏结果为标准，则噬菌体生物扩增法测定利福平耐药的敏感性为93-3％，特异性为87．9％，阴性预测值为96．4％，阳性预测值为78．9％，准确性为89．7％，涂阳等级（1＋～3＋）与实验的有效性相关。结论噬菌体生物扩增法测定痰标本利福平的耐药性只需2d，可作为快速筛诜方法府用千临床．     

微生物来源的Aurora—B激酶抑制剂的分离、结构鉴定和抗肿瘤活性研究

目的从微生物代谢产物中分离和纯化Aurora—B激酶抑制剂并检测其抗肿瘤活性。方法以野生型酵母菌Y300和ipZl-321温度敏感型突变株为模式菌跟踪活性组分;从阳性放线菌107A-01038发酵产物中分离纯化活性化合物并进行结构鉴定;体外酶学实验验证阳性化合物对Aurora—B激酶的抑制活性;MTT法检测阳性化合物对肿瘤细胞增殖的抑制活性;AnnexinV-FITC／PI双染法检测活性化合物对肿瘤细胞早期凋亡的诱导作用。结果从阳性放线菌107A-01038发酵产物中得到活性化合物酒渣碱甲酯（flazinmethylester）,酒渣碱甲酯对咖11．321突变株具有特异性抑制作用,其在野生型酵母菌株Y300和突变株ipl1—321的Ic50分别为48μmol／L和24μmol／L;体外酶学实验证实其对Aurora—B激酶具有抑制作用,其IC50为10μmol／L（ATP=50gmol／L）：酒渣碱甲酯对HepG2、A549、Hela肿瘤细胞均具有杀伤活性,IC50分别为13、11和10μmol／L。并能够诱导Hela细胞发生早期凋亡。结论得到-个微生物来源的具有抗肿瘤活性的Aurora—B激酶抑制剂-酒渣碱甲酯。     

Gamma interferon reverses inhibition of leukocyte bactericidal activity by a 25-kilodalton fraction from Mycobacterium tuberculosis.

In this study we examined the effects of Mycobacterium tuberculosis cell extracts on the phagocytic activity of polymorphonuclear leukocytes and cultured peripheral blood monocytes. M. tuberculosis cell extracts were fractionated on Sephacryl S-200 columns, and a 25-kilodalton glycolipoprotein was shown to inhibit the intracellular killing ability of these leukocytes but had no effect on their phagocytic potential. This same fraction inhibited fusion of phagosomes with lysosomes, as assessed by noting the transfer of acridine orange from lysosomes to phagosomes. This fraction was shown to have a maximal inhibitory effect when it was in the form of an intact carbohydrate-lipid-protein complex. Gamma interferon (IFN-gamma), but not IFN-alpha, reversed the inhibitory effect of the mycobacterial component on bactericidal activity and on fusion of phagosomes and lysosomes. Thus, this 25-kilodalton fraction of M. tuberculosis cell extract may be important in protecting organisms against phagocytic degradation, an effect which can be reversed by IFN-gamma.     

Antimicrobial activity of selected South African medicinal plants

ABSTRACT: BACKGROUND: Nearly 3,000 plant species are used as medicines in South Africa, with approximately 350 species forming the most commonly traded and used medicinal plants. In the present study, twelve South African medicinal plants were selected and tested for their antimicrobial activities against eight microbial species belonging to fungi, Mycobacteria, Gram-positive and Gram-negative bacteria. METHODS: The radiometric respiratory technique using the BACTEC 460 system was used for susceptibility testing against Mycobacterium tuberculosis, and the liquid micro-broth dilution was used for other antimicrobial assays. RESULTS: The results of the minimal inhibitory concentration (MIC) determinations indicated that the methanol extracts from Acacia karoo, Erythrophleum lasianthum and Salvia africana were able to prevent the growth of all the tested microorganisms. All other samples showed selective activities. MIC values below 100μg/ml were recorded with A. karoo, C. dentate, E. lasianthum, P. obligun and S. africana on at least one of the nine tested microorganisms. The best activity (MIC value of 39.06μg/ml) was noted with S. africana against E. coli, S. aureus and M. audouinii, and Knowltonia vesitoria against M. tuberculosis. CONCLUSION: The overall results of the present work provide baseline information for the possible use of the studied South African plant extracts in the treatment of microbial infections.     

Screening for HIV protease inhibitors by protection against activity-mediated cytotoxicity in Escherichia coli

Expressed retroviral proteases are often cytotoxic to the hosts. The cytotoxicity of a tethered dimer HIV protease described previously is particularly severe that transformed Escherichia coli cells could not survive the bactericidal activity of the low-level protease produced under uninduced conditions. The presence of HIV protease inhibitors protected the transformed cells from cytotoxic effects and allowed the growth of these cells on plates and in broth. A high throughput screening method was developed to seek compounds that served as "growth factors" for the HIV protease restricted cells. Several compounds identified by this screening supported the growth of these cells, preserved their viability, and inhibited HIV protease. This assay could be used as a general method for screening for inhibitors of recombinant enzymes that produce a cytotoxic phenotype in host cells.     

Simultaneous identification of Mycobacterium genus and Mycobacterium tuberculosis complex in clinical samples by 5'-exonuclease fluorogenic PCR

Early diagnosis of tuberculosis and screening of other mycobacteria is required for the appropriate management of patients. We have therefore developed a 5'-exonuclease fluorogenic PCR assay in a single-tube balanced heminested format that simultaneously detects Mycobacterium tuberculosis complex (MTC) and members of the Mycobacterium genus (MYC) using the 16S ribosomal DNA target directly on clinical samples. One hundred twenty-seven clinical samples (65 smear negative and 62 smear positive) with a positive culture result from 127 patients were tested, including 40 negative control specimens. The finding of both a positive MTC and probe value and a positive MYC probe value confirmed the presence of MTC or mycobacteria with a 100% positive predictive value. However, a negative value for MTC or MYC did not discount the presence of mycobacteria in the specimen. Interestingly, the addition of the MYC probe allowed the diagnosis of an additional 7% of patients with tuberculosis and rapid screening of nontuberculous mycobacteria (NTM). Thus, over 75% of the patients were diagnosed with mycobacterial disease by PCR. The sensitivity was much higher on smear-positive samples (90.3%) than smear-negative samples (49.2%) and was slightly higher for MTC than NTM samples. With regard to the origin of the sample, MTC pulmonary samples gave better results than others. In conclusion, we believe this test may be useful for the rapid detection of mycobacteria in clinical samples and may be a valuable tool when used together with conventional methods and the clinical data available.