LRET-based HT Screening:Inhibitors of Transcription(RMI)

描述(由申请人提供)：Sigma因子与核心RNA聚合酶的结合对于真细菌中特定的转录启动是必不可少的，因此对细胞生长至关重要。由于负责的蛋白结合区在所有真细菌中高度保守，但与真核RNA聚合酶有很大的不同，因此它是一个有希望的药物发现目标。本实验室利用Eu标记的Sigma70和IC5标记的RNA聚合酶(氨基酸残基100-309)的β亚基片段，建立了基于发光共振能量转移(LRET)的Sigma与RNA聚合酶(E.Coli)结合的均相分析方法。通过减少IC5的排放，通过LRET的损失来衡量Sigma结合的抑制。LRET提供的技术进步导致了一种非常可靠的适合高通量筛选的方法，并成功地用于筛选天然粗品文库。我们希望改进这一检测方法，开发适当的反筛选以最大限度地减少假阳性的数量，并开发进一步的方法来表征确认的命中。然后，我们将使用这些分析方法来筛选更大的文库，并有望识别和表征可能代表新抗生素药物开发的先导化合物的化合物。

DESCRIPTION (provided by applicant): The binding of sigma factors to core RNA polymerase is essential for specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, it is a promising target for drug discovery. A homogeneous assay for sigma-binding to RNA polymerase (E. coli) based on luminescence resonance energy transfer (LRET) has been developed in our lab using an europium-labeled sigma70 and IC5-labeled fragment of the beta-prime subunit of RNA polymerase (amino acid residues 100-309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5-emission.The technical advances offered by LRET resulted in a very robust assay suitable for a high-throughput screening and was successfully used to screen a crude natural product library. We would like to refine this assay, to develop appropriate counter-screens to minimize the number of false positives, and to develop further methods for characterizing the confirmed hits. Then we will use these assays to screen much larger libraries and hopefully to identify and characterize chemical compounds that might represent lead compounds for new antibiotic drug development.