High-throughput screening of RNA polymerase inhibitors using a fluorescent UTP analog
High-throughput screening of RNA polymerase inhibitors using a fluorescent UTP analog
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DOI：
10.1177/1087057106291978
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发表时间：
2006-12-01
影响因子：
--
通讯作者：
Roy, Raman K.
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文献类型：
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作者：
Bhat, Jyothi;Rane, Rajendra;Roy, Raman K.
RNA聚合酶（RNAP）是开发抗菌和抗结核药物的有效靶点。由于从致病性分枝杆菌中纯化大量天然RNA聚合酶是危险和繁琐的，因此使用大肠杆菌RNAP进行初步筛选。作者开发了一种高通量筛选（HTS）试验来筛选新型RNAP抑制剂。在该测定中，使用UTP的荧光类似物γ-氨基萘磺酸（γ-AmNS）UTP作为核苷酸底物之一。UMP在RNA中的掺入导致γ-AmNS-PPi的释放，其具有比（γ-AmNS）UTP更高的内在荧光。该测定在384孔格式中进行了优化，并用于筛选浓度为10 μ M的670，000种化合物。约0.1%的化合物对原代HTS的抑制率超过60%。使用相同测定法测试剂量反应的所有主要活性物质的EC 50均低于100 μ M。使用E. coli RNAP在常规放射性测定中显示出与耻垢分枝杆菌RNAP相当的活性。针对牛分枝杆菌RNAP（其与结核分枝杆菌RNAP亚基具有> 99%的序列同一性）也证实了选择用于命中-先导优化的命中的活性。
RNA polymerase (RNAP) is a well-validated target for the development of antibacterial and antituberculosis agents. Because the purification of large quantities of native RNA polymerase from pathogenic mycobacteria is hazardous and cumbersome, the primary screening was carried out using Escherichia coli RNAP. The authors have developed a high-throughput screening (HTS) assay to screen for novel inhibitors of RNAP. In this assay, a fluorescent analog of UTP, gamma-amino naphthalene sulfonic acid (gamma-AmNS) UTP, was used as one of the nucleotide substrates. Incorporation of UMP in RNA results in the release of gamma-AmNS-PPi, which has higher intrinsic fluorescence than (gamma-AmNS) UTP. The assay was optimized in a 384-well format and used to screen 670,000 compounds at a concentration of 10 mu M. About 0.1% of the compounds showed more than 60% inhibition in the primary HTS. All the primary actives tested for dose response using the same assay had an EC50 below 100 mu M. Eighty percent of the primary HTS actives obtained using E. coli RNAP showed comparable activity against Mycobacterium smegmatis RNAP in the conventional radioactive assay. Activity of hits selected for the hit-to-lead optimization was also confirmed against Mycobacterium bovis RNAP which has > 99% sequence identity with Mycobacterium tuberculosis RNAP subunits.