Kaur Kawaljit, Wu Xiaoqing, Fields James K, Johnson David K, Lan Lan, Pratt Miranda, Somoza Amber D, Wang Clay C C, Karanicolas John, Oakley Berl R, Xu Liang, De Guzman Roberto N
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, United States of America.
Molecular Graphics and Modeling Laboratory and the Computational Chemical Biology Core, University of Kansas, Lawrence, Kansas, United States of America.
PLoS One. 2017 Apr 17;12(4):e0175471. doi: 10.1371/journal.pone.0175471. eCollection 2017.
The RNA-binding protein Hu antigen R (HuR) binds to AU-rich elements (ARE) in the 3'-untranslated region (UTR) of target mRNAs. The HuR-ARE interactions stabilize many oncogenic mRNAs that play important roles in tumorigenesis. Thus, small molecules that interfere with the HuR-ARE interaction could potentially inhibit cancer cell growth and progression. Using a fluorescence polarization (FP) competition assay, we identified the compound azaphilone-9 (AZA-9) derived from the fungal natural product asperbenzaldehyde, binds to HuR and inhibits HuR-ARE interaction (IC50 ~1.2 μM). Results from surface plasmon resonance (SPR) verified the direct binding of AZA-9 to HuR. NMR methods mapped the RNA-binding interface of HuR and identified the involvement of critical RNA-binding residues in binding of AZA-9. Computational docking was then used to propose a likely binding site for AZA-9 in the RNA-binding cleft of HuR. Our results show that AZA-9 blocks key RNA-binding residues of HuR and disrupts HuR-RNA interactions in vitro. This knowledge is needed in developing more potent AZA-9 derivatives that could lead to new cancer therapy.
RNA结合蛋白Hu抗原R（HuR）与靶标mRNA的3'非翻译区（UTR）中的富含AU元件（ARE）结合。HuR-ARE相互作用使许多在肿瘤发生中起重要作用的致癌mRNA稳定。因此，干扰HuR-ARE相互作用的小分子可能会抑制癌细胞的生长和进展。通过荧光偏振（FP）竞争试验，我们鉴定出源自真菌天然产物asperbenzaldehyde的化合物氮杂环庚三烯酮-9（AZA-9），它能与HuR结合并抑制HuR-ARE相互作用（半数抑制浓度约为1.2μM）。表面等离子体共振（SPR）结果证实了AZA-9与HuR的直接结合。核磁共振（NMR）方法绘制了HuR的RNA结合界面，并确定了关键RNA结合残基在AZA-9结合中的作用。然后利用计算对接提出AZA-9在HuR的RNA结合裂隙中的可能结合位点。我们的结果表明，AZA-9在体外阻断了HuR的关键RNA结合残基并破坏了HuR-RNA相互作用。开发更有效的AZA-9衍生物以带来新的癌症治疗方法需要这些知识。
