Application of a novel RNA-protein interaction assay to develop inhibitors blocking RNA-binding of the HuR protein.

Abstract

RNA-protein interactions play an important regulatory role in several biological processes. For example, the RNA-binding protein HuR (human antigen R) binds to its target mRNAs and regulates their translation, stability, and subcellular localization. HuR is involved in the pathogenic processes of various diseases. Thus, small molecules blocking RNA-binding of HuR may be useful in a variety of diseases. Previously, we identified STK018404 as a small molecule targeting the HuR-RNA interaction. Based on this study we identified optimized compounds by exploiting combined structure-based and ligand-based computational approaches. To test a series of these compounds, we developed a novel readout system for the HuR-RNA interaction. Traditional methods to detect RNA-protein interaction come with some disadvantages: they require significant reagent optimization and may be difficult to optimize for weakly expressed RNA molecules. The readout often requires amplification. Thus, these methods are not well suited for quantitative analysis of RNA-protein interactions. To achieve an easy-to-perform, rapid, and robust detection of RNA-protein binding, we applied a split luciferase reporter system, to detect the interaction between HuR and its target RNA. We expressed one luciferase fragment as a fusion protein with HuR. The second luciferase fragment was Streptavidin-coated and coupled to a biotinylated RNA-oligo comprising an AU-rich HuR-binding element. The binding between HuR and its target RNA-oligo then allowed reconstitution of the functional luciferase that was detectable by luminescence. Using the split luciferase reporter system, we present here a series of optimized compounds that we developed.