Aberrant expression of microRNA-21 (miR-21) is recognized as a key determinant in cancer progression through dysregulated post-translational control of several tumor suppressor genes. Hence, identification of compounds directed against miR-21 is representing an emerging opportunity for the development of candidate drugs with innovative modalities, such as ribotacs or interferents with pre-miR-21 maturation. Here, we report the identification of compounds targeting pre-miR-21 through screening of small-molecule collections and using differential scanning fluorimetry (DSF) as biophysical binding assay.

DSF monitors the ability of small molecules to engage RNA through increased thermal stability. The assay was developed using a hairpin-structured pre-miR-21 and a fluorogenic RNA-binding dye. Compound-induced thermal shift of pre-miR-21 was detected by QuantStudio® RT-qPCR system in 384 well plate format. Reference compounds, as well as single-point pre-miR-21 mutant and equivalent DNA sequences were assessed to validate the assay selectivity. Screening was performed on an unbiased collection of 1300 biologically active compounds and on a focused library of 2200 compounds enriched for RNA binding moieties.

The DSF assay was optimized to achieve a screening-suited performance while enhancing its sensitivity towards putative RNA-binding molecules. Selectivity was proven by using as positive control dovitinib, a pre-miR-21 binding molecule that inhibits miR-21 biogenesis. The assay displayed very high robustness, with RZ’ of 0.83 and CV% of 0.7. Combined screening of unbiased and focused libraries of 3500 compounds in total identified 10 primary hits (0.3% hit rate) triggering a significant Tm shift for pre-miR-21. Primary hits were triaged at increasing concentration, with 8 compounds confirmed to display dose-dependent saturation curves with aKD values in the 1-/2-digit micromolar range. Some chemical scaffolds of confirmed hits displayed similarity with biologically active compounds known to target nucleic acids, while others appeared to be new structures. A subset showed selective binding to pre-miR-21 over other ribonucleotides. In all cases, confirmed hits are novel in the pre-miR-21 binding mechanism, and follow-up studies are currently ongoing with orthogonal biophysical assays, as well as on cancer cell systems to confirm their ability to interfere with pre-miR-21 processing.

The combination of DSF and screening has identified novel series of pharmacophores acting as pre-miR-21 binders, with the potential to serve as building blocks for the development of lead compounds offering an unconventional modality to target cancer progression.