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

描述（由申请人提供）：sigma因子与核心RNA聚合酶的结合是真细菌特异性转录起始的必要条件，因此对细胞生长至关重要。由于负责的蛋白质结合区在所有真核细菌中高度保守，但与真核RNA聚合酶有很大不同，因此它是药物发现的一个有希望的靶点。我们的实验室利用铕标记的sigma70和ic5标记的RNA聚合酶β -引物亚基片段（氨基酸残基100-309）开发了一种基于发光共振能量转移（LRET）的sigma与RNA聚合酶（大肠杆菌）结合的均匀分析方法。通过ic5释放减少LRET的损失来测量sigma结合的抑制作用。LRET提供的技术进步导致了适合高通量筛选的非常稳健的分析，并成功地用于筛选粗天然产物库。我们希望改进这一检测方法，开发适当的反筛选，以尽量减少假阳性的数量，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来筛选更大的文库，并希望识别和表征可能代表新抗生素药物开发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.