Development and validation of a high-density fluorescence polarization-based assay for the trypanosoma RNA triphosphatase TbCet1.
Development and validation of a high-density fluorescence polarization-based assay for the trypanosoma RNA triphosphatase TbCet1.
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基于锥虫RNA三磷酸酶TBCET1的高密度荧光极化测定的开发和验证。
DOI：
10.2174/138620709787581729
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发表时间：
2009-03
影响因子：
1.8
通讯作者：
Djaballah H
中科院分区：
文献类型：
--
作者：
Antczak C;Shum D;Radu C;Seshan VE;Djaballah H
RNA三磷酸酶是开发广谱抗原虫、抗病毒和抗真菌药物的有吸引力且大多未被探索的治疗靶点。孔雀石绿作为磷酸酶读数的使用具有很好的特性和广泛的应用。然而，该反应依赖于产生大量的无机磷，这使得该方法不容易在1536孔格式中进行筛选。所需的过长的阅读时间也禁止使用它来筛选大型化学库。为了克服这些限制，我们试图开发一种基于荧光偏振(FP)的三磷酸酶分析方法，与小型化和快速读数兼容。为此，我们利用了这类酶的核苷三磷酸酶活性，成功地使TranscreenerRNAADP分析方法基于免疫竞争荧光偏振检测生成的腺苷二磷酸与1536well格式的™三磷酸酶TbCet1相适应。我们还在3000种化合物的初步筛选中测试了这种新开发的分析方法的性能，并确认了获得的HITS的活性。我们提出并讨论了我们的发现及其对通过高通量筛选发现新药的重要性。
RNA triphosphatases are attractive and mostly unexplored therapeutic targets for the development of broad spectrum antiprotozoal, antiviral and antifungal agents. The use of malachite green as a readout for phosphatases is well characterized and widely employed. However, the reaction depends on high quantities of inorganic phosphate to be generated, which makes this assay not easily amenable to screening in 1536-well format. The overly long reading times required also prohibit its use to screen large chemical libraries. To overcome these limitations, we sought to develop a fluorescence polarization (FP) -based assay for triphosphatases, compatible with miniaturization and fast readouts. For this purpose, we took advantage of the nucleoside triphosphatase activity of this class of enzyme to successfully adapt the Transcreener™ ADP assay based on the detection of generated ADP by immunocompetition fluorescence polarization to the RNA triphosphatase TbCet1 in 1536-well format. We also tested the performance of this newly developed assay in a pilot screen of 3,000 compounds and we confirmed the activity of the obtained hits. We present and discuss our findings and their importance for the discovery of novel drugs by high-throughput screening.
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影响因子：
5.8
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作者：
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