A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P.
A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P.
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DOI：
10.1093/nar/gku850
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发表时间：
2014-11-10
影响因子：
14.9
通讯作者：
Fierke CA
中科院分区：
文献类型：
--
作者：
Liu X;Chen Y;Fierke CA
核糖核酸酶P （RNase P）是催化前体tRNA (pre-tRNA) 5 '端成熟的重要内切酶。细菌RNase P是一个有吸引力的潜在抗菌靶标，因为它是细胞存活所必需的，与真核对应物相比，它具有独特的亚基组成。为了加速RNase P的结构-功能研究和抑制剂的发现，我们利用荧光偏振/各向异性（FP/FA）和5 '端荧光素标记的预trnaasp底物开发了第一个实时RNase P活性测定。这种FP/FA分析也检测小分子与前trna的结合。新霉素B和卡那霉素B与pre-tRNAAsp结合的Kd值与其抑制RNase P的IC50值相当，表明这些抗生素与pre-tRNA底物结合有助于抑制活性。该实验对高通量筛选（HTS）进行了优化，以从2880化合物文库中鉴定特异性RNase P抑制剂。一种天然产物衍生物——六乙酸油松醇被鉴定为枯草芽孢杆菌RNase P的新抑制剂。本文报道的FP/FA方法和抑制剂将进一步加深我们对RNase P分子识别的理解，并有助于发现针对RNase P的抗菌化合物。
Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the 5′ end maturation of precursor tRNA (pre-tRNA). Bacterial RNase P is an attractive potential antibacterial target because it is essential for cell survival and has a distinct subunit composition compared to the eukaryal counterparts. To accelerate both structure-function studies and discovery of inhibitors of RNase P, we developed the first real-time RNase P activity assay using fluorescence polarization/anisotropy (FP/FA) with a 5′ end fluorescein-labeled pre-tRNAAsp substrate. This FP/FA assay also detects binding of small molecules to pre-tRNA. Neomycin B and kanamycin B bind to pre-tRNAAsp with a Kd value that is comparable to their IC50 value for inhibition of RNase P, suggesting that binding of these antibiotics to the pre-tRNA substrate contributes to the inhibitory activity. This assay was optimized for high-throughput screening (HTS) to identify specific inhibitors of RNase P from a 2880 compound library. A natural product derivative, iriginol hexaacetate, was identified as a new inhibitor of Bacillus subtilis RNase P. The FP/FA methodology and inhibitors reported here will further our understanding of RNase P molecular recognition and facilitate discovery of antibacterial compounds that target RNase P.
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影响因子：
3.2
作者：
Gruegelsiepe, H;Willkomm, DK;Hartmann, RK
通讯作者：
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影响因子：
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