共查询到19条相似文献,搜索用时 93 毫秒
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药物筛选新方法--高通量筛选 总被引:7,自引:0,他引:7
介绍了高通量筛选的概念、原理及其各个组成部分,着重阐明了高通量筛选的筛选模式,检测方法和应用实例,并简单介绍了国内在这方面的研究进展。 相似文献
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邓志婷戚欣李静 《现代生物医学进展》2012,12(6):1176-1178
目前的新药研发策略已从传统的以活性为主导的筛选模式,转变为在测定药效的同时平行评价化合物的ADMET性质,并建立了系列体外ADMET高通量筛选技术。本文综述了近年来药物体外ADMET高通量筛选技术的研究进展。 相似文献
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为发现TRPV3调节剂,通过荧光成像分析系统检测钙浓度,建立高通量筛选瞬时受体势V3通道(Transient receptor potential V3,TRPV3)调节剂的细胞模型.将TRPV3表达载体转染人胚肾(HEK-293)细胞,抗生素筛选稳定表达TRPV3的细胞系,选取TRPV3特异性调节剂作用于细胞模型,应用荧光成像分析系统测钙实验检测TRPV3高表达细胞系药理学特征,同时优化实验条件,考察模型的稳定性,并评估应用于96孔板及384孔板进行高通量筛选的可靠性及准确性.获得了高表达TRPV3的HEK-293稳定细胞系,通过TRPV3离子通道调节的钙流信号与TRPV3特异性调节剂成剂量依赖关系,优化得到了最适筛选条件,该模型稳定,灵敏,通过Z因子及Spiking检测,完全符合高通量筛选需求.利用此细胞模型通过检测钙信号可筛选TRPV3调节剂. 相似文献
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为了建立黑色素皮质素受体(MC4R)激动剂的高通量筛选方法,将人的MC4R基因质粒(hMC4R/pCDNA3.1)与报告基因质粒(3×CRE/3×MRE/SRE-LUC)按1∶5的比例共转染到HEK293细胞,通过G418筛选,建立了稳定的MC4R激动剂筛选细胞株.利用MC4R内源激动剂α-MSH探索和优化了每孔接种细胞数目、激动剂孵育时间、溶剂DMSO终浓度、荧光素酶底物浓度等筛选条件,建立了可靠的筛选方法.实验表明:当细胞数目为4×104个/孔,激动剂孵育时间为8 h,每孔DMSO终浓度小于1%和α-MSH终浓度为1 μmol/L时,系统Z'-因子接近0.7,能够用于MC4R激动剂的高通量筛选. 相似文献
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作为先进的细胞电生理技术,膜片钳一直被奉为研究离子通道的“金标准”。应用膜片钳技术可以证实细胞膜上离子通道的存在并能对其电生理特性、分子结构、药物作用机制等进行深入的研究。基因组学、蛋白质组学研究表明,以离子通道为靶标的药物研究在未来具有很大的发展空间。为了突破由于筛选技术所造成的针对离子通道为靶标的药物研发的瓶颈,近年来,对膜片钳技术进行了改进以适合药物高通量筛选的需求,由此产生了一些新的技术。本文就最近几年膜片钳技术的新进展及其在药物高通量筛选中的应用进行了综述。 相似文献
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新药研发过程中.通过筛选而获得具有生物活性的先导化合物.是创新药物研究的关键.目前药物筛选模型已经从传统的整体动物、器官和组织水平发展到细胞和分子水平。创新药物的发现都离不开采用适当的药物作用靶点对大量化合物样品进行筛选.而且筛选规模越大,发现新药的机会就越多。随着计算机技术、生物芯片、蛋白质组学、组合化学等的发展.高通量药物筛选技术应运而生。高通量筛选体系在创新药物筛选中的应用是新药开发研究的一个重要领域。 相似文献
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Donovan RS Datti A Baek MG Wu Q Sas IJ Korczak B Berger EG Roy R Dennis JW 《Glycoconjugate journal》1999,16(10):607-615
Glycosyltransferases mediate changes in glycosylation patterns which, in turn, may affect the function of glycoproteins and/or glycolipids and, further downstream, processes of development, differentiation, transformation and cell-cell recognition. Such enzymes, therefore, represent valid targets for drug discovery. We have developed a solid-phase glycosyltransferase assay for use in a robotic high-throughput format. Carbohydrate acceptors coupled covalently to polyacrylamide are coated onto 96-well plastic plates. The glycosyltransferase reaction is performed with recombinant enzymes and radiolabeled sugar-nucleotide donor at 37°C, followed by washing, addition of scintillation counting fluid, and measurement of radioactivity using a 96-well -counter. Glycopolymer construction and coating of the plastic plates, enzyme and substrate concentrations, and linearity with time were optimized using recombinant Core 2 1-6-N-acetylglucosaminyltransferase (Core 2 GlcNAc-T). This enzyme catalyzes a rate-limiting reaction for expression of polylactosamine and the selectin ligand sialyl-Lewisx in -glycans. A glycopolymer acceptor for 1-6-N-acetylglucosaminyltransferase V was also designed and shown to be effective in the solid-phase assay. In a high-throughput screen of a microbial extract library, the coefficient of variance for positive controls was 9.4%, and high concordance for hit validation was observed between the Core 2 GlcNAc-T solid-phase assay and a standard solution-phase assay. The solid-phase assay format, which can be adapted for a variety of glycosyltransferase enzymes, allowed a 5–6 fold increase in throughput compared to the corresponding solution-phase assay. 相似文献
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帕金森病是一种常见的神经系统退行性疾病。多巴脱羧酶(DDC)是帕金森病研究的靶点蛋白之一,但是目前没有高通量的测活模型。因此,需要构建一种高通量多巴脱羧酶抑制剂的筛选模型,用于发现新型抑制剂。采用克隆表达纯化得到多巴脱羧酶和用于酶偶联反应的磷酸烯醇式丙酮酸羧化酶(PEPC)。基于一系列酶联反应将CO2固定,检测其含量,从而测定多巴脱羧酶的活性。结果得到人源多巴脱羧酶和磷酸烯醇式丙酮酸羧化酶的体外纯酶,建立了一种高通量筛选模型,并且从70个天然化合物中,筛选得到2个多巴脱羧酶的抑制剂。成功构建了一种基于体外纯酶高通量多巴脱羧酶抑制剂的筛选模型。 相似文献
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Wang MW Hao X Chen K 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2007,362(1482):1093-1105
Natural products have been applied to human healthcare for thousands of years. Drug discovery in ancient times was largely by chance and based on clinical practices. As understanding of therapeutic benefits deepens and demands for natural products increase, previously serendipitous discoveries evolve into active searches for new medicines. Many drugs presently prescribed by physicians are either directly isolated from plants or are artificially modified versions of natural products. Scientists are looking for lead compounds with specific structures and pharmacological effects often from natural sources. Experiences and successes of Chinese scientists in this specialized area have resulted in a number of widely used drugs. The tremendous progress made in life sciences has not only revealed many pathological processes of diseases, but also led to the establishment of various molecular and cellular bioassays in conjunction with high-throughput technologies. This is advantageous and permits certain natural compounds that are difficult to isolate and purify, and compounds that are difficult to synthesize, to be assayed. The transition from traditional to empirical and to molecular screening will certainly increase the probability of discovering new leads and drug candidates from natural products. 相似文献
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A high-throughput fluorescent polarization assay for nuclear receptor binding utilizing crude receptor extract. 总被引:2,自引:0,他引:2
Siqi Lin Cindy L Bock Debra B Gardner Jeffery C Webster Margaret F Favata James M Trzaskos Kevin R Oldenburg 《Analytical biochemistry》2002,300(1):15-21
A homogenous high-throughput assay has been developed to measure the binding between nuclear receptors and test compounds. This assay applies a fluorescence polarization (FP) detection method using human glucocorticoid receptor (GR) as a model system. Crude receptor extract, which requires no additional purification, is used in the assay. The binding conditions (i.e., DMSO tolerance, temperature, stability, and variability) have been investigated and validated. At the optimized conditions, a signal-to-background ratio of 2:1 and a Z'-factor of 0.7 was achieved in a 384-well format. Several known strong and weak GR ligands have been evaluated in this system. Possible interference of fluorescent compounds and methods to identify false positives are also discussed. This FP-based assay system can potentially be used for many soluble nuclear receptors in high-throughput binding assays. 相似文献
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基于新一代高通量测序的环境微生物转录组学研究进展 总被引:3,自引:0,他引:3
环境微生物转录组学是一门新兴学科,它以复杂环境样品中的微生物mRNA为研究对象,利用近年兴起的RNA-Seq高通量测序技术,在整体水平上对环境微生物的基因表达水平和调控规律进行研究.本文概述了环境微生物转录组研究从样品的采集保存、RNA提取、mRNA的富集、cDNA合成直到高通量测序及数据分析的基本流程.总结了该技术面临的主要瓶颈:环境样品mRNA含量低、腐植酸等干扰杂质多、rRNA去除程度有限.针对RNA的提取、纯化以及mRNA的富集这些重点步骤,详细阐述了近年来在提高mRNA的得率与纯度上的方法学进展.重点介绍了高通量测序数据的处理及分析方法,从测序数据的质量控制、序列组装、rRNA的鉴定及去除、功能基因注释及分类到差异表达基因的鉴定.最后总结了近年来环境微生物转录组学在新基因的发现、不同环境条件下微生物的基因表达及调控规律研究、有机物的代谢路径分析等3个主要研究领域的广泛应用.随着测序技术及生物信息学分析工具的发展进步,环境微生物转录组学将具有更广阔的应用前景. 相似文献
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核质运输是真核细胞的重大基本生命活动。核质运输小分子抑制剂不仅可以广泛应用并促进核质运输相关的基础研究,同时也为相关疾病、尤其是病毒性疾病的药物开发提供有利线索。然而,目前针对核质运输的商业化小分子仅有Leptomycin B一种。建立一个针对整个核质运输通路的小分子筛选平台,将有利于筛选与获得多种干扰核质运输的小分子。文章利用NZGFP和CZGFP可以重组为具有荧光GFP的特性,构建NZGFP-NES和CZGFP-NLS,将NZGFP和CZGFP分别定位在细胞质与细胞核中;当核内运或核外运通路被干扰,NZGFP和CZGFP定位发生改变并聚集重组为具有荧光的GFP。该方法可以有效检测核外运小分子抑制剂LeptomycinB的作用,为针对整个核质运输通路的高通量小分子筛选提供了一个有效平台。 相似文献
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《Expert review of proteomics》2013,10(1):111-121
The biological significance of protein interactions, their method of generation and reliability is briefly reviewed. Protein interaction networks adopt a scale-free topology that explains their error tolerance or vulnerability, depending on whether hubs or peripheral proteins are attacked. Networks also allow the prediction of protein function from their interaction partners and therefore, the formulation of analytical hypotheses. Comparative network analysis predicts interactions for distantly related species based on conserved interactions, even if sequences are only weakly conserved. Finally, the medical relevance of protein interaction analysis is discussed and the necessity for data integration is emphasized. 相似文献