Design and synthesis of small molecules targeting pre-miRNA21 toward the development of Ribonuclease Targeting Chimeras (RIBOTACs) to regulate the oncogenic miRNA21

RNA biology has undergone a major transformation and now stands at the forefront of modern drug discovery, enabling the development of innovative therapeutics. Despite this progress, the rational design of small molecules that selectively target structured RNAs remains challenging because RNA differs fundamentally from classical protein targets in its folding, dynamics, and recognition principles. These characteristics raise important questions regarding RNA druggability and require tailored discovery strategies. Furthermore, the identification of new chemical entities for RNA targeting is limited by the scarcity of well-validated targets. Among these, microRNA-21 (miR-21) represents a therapeutically relevant and structurally complex model. As one of the earliest discovered mammalian miRNAs, miR-21 plays key roles in the onset and progression of several diseases. Consequently, the development of new chemical entities targeting pre-miR-21, the precursor of mature miR-21, constitutes a highly validated approach that may benefit from emerging therapeutic modalities. The drug discovery toolbox has expanded considerably over the last decade, providing medicinal chemists with access to diverse chemical modalities and broader chemical space to modulate challenging targets. Among these innovations, Ribonuclease Targeting Chimeras (RIBOTACs) represent a promising strategy that exploits endogenous RNase L to selectively degrade disease-associated RNAs. Based on these premises, this thesis aimed to develop new strategies for miR-21 drug discovery through two complementary projects. A reported NMR fragment-screening approach was leveraged within a fragment-based drug design framework to identify novel chemotypes targeting structured regions of pre-miR-21. Hits were validated through native mass spectrometry, biochemical and biophysical assays, and pharmacological studies, leading to selective ligands active in a neuroblastoma cell model. The most promising binders were converted into pre-miR-21 RIBOTACs to promote RNA degradation through RNase L recruitment. Preliminary results support the proposed mechanism of action and highlight the potential of this approach for RNA-targeted therapeutics.