An important paradigm in current cancer therapeutics is the progressive shift from broad-spectrum chemotherapy to personalized, targeted treatment. Following intense research, a few proteins have been proposed as neuroblastoma-specific targets. The two LIN28 paralog RNA-binding proteins (RBPs) are well known oncogenic drivers in several types of tumors; they exert their effects mainly by preventing maturation of miRNAs of the let-7 family, which drives cell differentiation. In neuroblastoma LIN28B is of particular importance. The first genome-wide association study for familiar neuroblastoma unveiled a polymorphism closely associated with disease and survival influencing LIN28B expression. Moreover, LIN28B is extensively overexpressed in NB compared to several other tumor types, and its forced expression in the sympathoadrenergic lineage of mice reproduces the human disease.
We aimed at preclinically identifying small molecules able to interfere with the binding of LIN28B to the pre-let-7 miRNAs, therefore increasing expression of the let-7 miRNAs and promoting differentiation of neuroblastoma with potential therapeutic outcomes. To this aim, we exploited the amplified luminescent proximity homogeneous assay technology. A human LIN28B recombinant protein was produced and challenged with a 5’-biotinylated ssRNA corresponding to the pre-let-7g pre-miRNA. We demonstrated a sequence-specific, high-affinity interaction between the RNA and the LIN28B RBP, the coefficient of variation, the Z-factor value, and the signal-to-background ratio, that collectively indicated the robustness and reliability of the assay in a high-throughput format. Then we applied the assay to a collection of ~2000 small molecules with known bioactivity. From the primary screening we selected 80 hits, and after validation and counter-screening we obtained 30 molecules, some of which characterized by a common chemical scaffold.
Docking in silico experiments followed by medicinal chemistry allowed us to design a second, focused small molecule library, which was tested in soaking with LIN28B protein crystals. The selected molecules were finally subjected to RNA Electrophoretic Mobility Shift Assay and a number of phenotypic studies by high content analysis.