Integrated in silico and experimental screening identifies novel ligands that target precursor microRNA-31 at the Dicer cleavage site

Abstract

MicroRNAs (miRNAs) regulate gene expression and the dysregulation in mature miRNA levels has been implicated in a wide variety of diseases. In particular, altered levels of mature microRNA-31 (miR-31) has been linked to a variety of different cancers. Targeting functionally relevant sites of the precursor structure of miR-31 with small molecules offer a strategy to regulate miR-31 maturation. Herein we describe a virtual screening approach to explore the druggability of the precursor structure of microRNA-31 (pre-miR-31). We used a structure-guided approach to virtually screen a fragment library and followed up with experimental characterization of top-ranking candidates, leading to the identification of several compounds that bound to pre-miR-31. Further characterization of the RNA-ligand complexes by heteronuclear single quantum coherence (HSQC) NMR spectroscopy revealed three compounds bound pre-miR-31 at the Dicer cleavage site, suggesting that these compounds may function to inhibit Dicer processing. Using these initial hits, we performed chemical structure similarity searches and identified additional binders of pre-miR-31 that had equivalent or enhanced binding relative to the parent compounds. These studies suggest a generalizable approach by which RNA-binding ligands can be identified from large chemical databases. These hits can then be further optimized to improve affinity and specificity for downstream functional assays.