Fukuzumi Takeo, Murata Asako, Aikawa Haruo, Harada Yasue, Nakatani Kazuhiko
Department of Regulatory Bioorganic Chemistry, The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047 (Japan).
Chemistry. 2015 Nov 16;21(47):16859-67. doi: 10.1002/chem.201502913. Epub 2015 Oct 6.
The metabolic stream of microRNA (miRNA) production, the so-called maturation process of miRNAs, became one of important metabolic paths for drug-targeting to modulate the expression of genes related to a number of diseases. We carried out discovery studies on small molecules binding to the precursor of miR-29a (pre-miR-29a) from a chemical library containing 41,119 compounds (AQ library) by the fluorescent indicator displacement (FID) assay using the xanthone derivative X2SdiMe as a fluorescent indicator. The FID assay provided 1075 compounds, which showed an increase of fluorescence. These compounds were subsequently submitted to a binding analysis in a surface plasmon resonance (SPR) assay on a pre-miR-29a immobilized surface. 21 hit compounds were identified with a good reproducibility in the binding. These compounds have not been reported to bind to RNA until now and can be classified into two groups on the basis of the kinetics in the binding. To gain more information on the motif structures that could be necessary for the binding to pre-miR-29a, 19 substructures were selected from the hit compounds. The substructure library (SS library) which consisted of 362 compounds was prepared from the AQ library. An SPR assay of the SS library on pre-miR-29a-immobilized surface suggested that five substructures could potentially be important structural motifs to bind to pre-miR-29a. These studies demonstrate that the combination of FID-based screening of chemical library and subsequent SPR assay would be one way for obtaining practical solutions for the discovery of molecules which bind to the target pre-miRNAs.
微小RNA（miRNA）产生的代谢流，即所谓的miRNA成熟过程，成为药物靶向调控与多种疾病相关基因表达的重要代谢途径之一。我们通过使用呫吨酮衍生物X2SdiMe作为荧光指示剂的荧光指示剂置换（FID）测定法，对来自包含41119种化合物的化学文库（AQ文库）中与miR-29a前体（pre-miR-29a）结合的小分子进行了发现研究。FID测定法提供了1075种显示荧光增加的化合物。随后将这些化合物在固定有pre-miR-29a的表面上进行表面等离子体共振（SPR）测定法的结合分析。鉴定出21种命中化合物，其结合具有良好的重现性。这些化合物迄今为止尚未见有与RNA结合的报道，并且根据结合动力学可分为两组。为了获得更多关于与pre-miR-29a结合可能必需的基序结构的信息，从命中化合物中选择了19个亚结构。由362种化合物组成的亚结构文库（SS文库）是从AQ文库制备的。在固定有pre-miR-29a的表面上对SS文库进行的SPR测定表明，五个亚结构可能是与pre-miR-29a结合的重要结构基序。这些研究表明，基于FID的化学文库筛选与随后的SPR测定相结合将是获得发现与目标前体miRNA结合的分子的实际解决方案的一种方法。
