Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction
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- 作者：Xiaoqing Wu ; Lan Lan ; David Michael Wilson ; Rebecca T. Marquez ; Wei-chung Tsao ; Philip Gao ; Anuradha Roy ; Benjamin Andrew Turner ; Peter McDonald ; Jon A Tunge ; Steven A Rogers ; Dan A. Dixon ; Jeffrey Aub茅 ; Liang Xu
- 刊名：ACS Chemical Biology
- 出版年：2015
- 出版时间：June 19, 2015
- 年：2015
- 卷：10
- 期：6
- 页码：1476-1484
- 全文大小：613K
- ISSN：1554-8937
文摘
HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3鈥?untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR鈥揂RE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of 鈭?000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR鈥揂RE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers.