Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Ludwika Pasteura 5, 02-093, Warsaw, Poland.
Centre of New Technologies, University of Warsaw, Stefana Banacha 2c, 02-097, Warsaw, Poland.
Sci Rep. 2021 Apr 8;11(1):7687. doi: 10.1038/s41598-021-87306-8.
Fluorescence anisotropy (FA) is a powerful technique for the discovery of protein inhibitors in a high-throughput manner. In this study, we sought to develop new universal FA-based assays for the evaluation of compounds targeting mRNA 5' cap-binding proteins of therapeutic interest, including eukaryotic translation initiation factor 4E and scavenger decapping enzyme. For this purpose, a library of 19 carboxyfluorescein probes based on 7-methylguanine nucleotides was evaluated as FA probes for these proteins. Optimal probe:protein systems were further investigated in competitive binding experiments and adapted for high-throughput screening. Using a small in-house library of compounds, we verified and confirmed the accuracy of the developed FA assay to study cap-binding protein binders. The applications of the most promising probes were then extended to include evaluation of allosteric inhibitors as well as RNA ligands. From this analysis, we confirmed the utility of the method to study small molecule ligands and evaluate differently 5' capped RNAs.
荧光各向异性（FA）是一种强大的技术，可用于高通量地发现蛋白质抑制剂。在这项研究中，我们试图开发新的基于 FA 的通用测定法，以评估针对治疗相关的 mRNA 5'帽结合蛋白的化合物，包括真核翻译起始因子 4E 和清道夫脱帽酶。为此，我们评估了基于 7-甲基鸟嘌呤核苷酸的 19 个羧基荧光素探针库作为这些蛋白质的 FA 探针。在竞争结合实验中进一步研究了最佳探针：蛋白质系统，并将其适应于高通量筛选。使用我们内部的小型化合物文库，我们验证并确认了所开发的 FA 测定法用于研究帽结合蛋白结合物的准确性。然后，将最有前途的探针的应用扩展到包括变构抑制剂以及 RNA 配体的评估。通过该分析，我们证实了该方法可用于研究小分子配体并评估不同的 5'加帽 RNA。
