The targeting of non-coding RNAs (nc-RNAs) appears as a promising frontier in current drug discovery, since their extended biological pathways are still largely unexplored and could indeed provide opportunities to modulate biological factors that are not targetable with traditional protein-based drug discovery. Within this context, micro-RNA-21 (miR21) appears as an attractive potential pharmaceutical target, due to its aberrant overexpression in various cancers and its well-established anti-apoptotic role. At IIT, our research focuses on modulating miR21 levels in cells by means of small molecule compounds, thereby establishing a platform for a broader drug discovery campaign. To achieve this objective, the modulation of miR21 biogenesis was investigated, identifying the precursor of miR21, i.e., pre-miR21, as the biological target of this medicinal chemistry project. Starting from a 19F-NMR-based screening of circa 600 low-molecular-weight compounds (fragments) (IIT), followed by a fluorescence-based testing of the preliminary hits for binding confirmation (University of Nice), a few initial hit compounds were identified with a promising affinity toward pre-miR21. Among these compounds, hit 1, featuring a pyrazolo[1,5-a]pyrimidine scaffold, was selected for its chemical tractability and further developed through a ligand-based medicinal chemistry campaign. During my research activity at IIT, I designed and synthesized 75 analogues of hit 1 with the aim of increasing the affinity of this chemotype toward pre-miR21. Biochemical activity was evaluated using the fluorescence assay developed in Dr. Duca’s laboratory, which guided the further evolution of this chemotype. During this process, a well-defined structure–activity relationship (SAR) was established, suggesting possible target engagement. Ligand–target interactions were further investigated during my research period at AstraZeneca (Sweden) through NMR structural studies. For achieving these outcomes, fluorinated probes were produced through solid-phase synthesis by systematically introducing them into the pre-miR21 structure. These studies provided insights into the RNA secondary structure and demonstrated that the synthesized compounds establish specific interactions with pre-miR21. Biochemical target engagement was also assessed using an NMR-based assay, providing orthogonal validation to the fluorescence-based method. SAR analysis established across both approaches showed consistent trends, thus confirming the putative modulation of miR21 by small molecule compounds featuring this novel chemotype. Having confirmed favourable biochemical ligand–target interactions, the cellular activity of the most promising novel compounds was evaluated by assessing modulation of both mature miR21 levels and related targets. The data produced demonstrated that the identified compounds are indeed active in PANC-1 cell-line and exhibit preliminary indications of possible target engagement. Overall, throughout this activity set-up, fragment-based drug discovery was shown to be effective from initial screening to hit evolution, yielding small molecules that remain amenable to further optimization and that may serve as starting points for a broader medicinal chemistry campaign.
Small molecule modulators of the activity of non-coding RNAs
QUAIA, RICCARDO
2026-06-29
Abstract
The targeting of non-coding RNAs (nc-RNAs) appears as a promising frontier in current drug discovery, since their extended biological pathways are still largely unexplored and could indeed provide opportunities to modulate biological factors that are not targetable with traditional protein-based drug discovery. Within this context, micro-RNA-21 (miR21) appears as an attractive potential pharmaceutical target, due to its aberrant overexpression in various cancers and its well-established anti-apoptotic role. At IIT, our research focuses on modulating miR21 levels in cells by means of small molecule compounds, thereby establishing a platform for a broader drug discovery campaign. To achieve this objective, the modulation of miR21 biogenesis was investigated, identifying the precursor of miR21, i.e., pre-miR21, as the biological target of this medicinal chemistry project. Starting from a 19F-NMR-based screening of circa 600 low-molecular-weight compounds (fragments) (IIT), followed by a fluorescence-based testing of the preliminary hits for binding confirmation (University of Nice), a few initial hit compounds were identified with a promising affinity toward pre-miR21. Among these compounds, hit 1, featuring a pyrazolo[1,5-a]pyrimidine scaffold, was selected for its chemical tractability and further developed through a ligand-based medicinal chemistry campaign. During my research activity at IIT, I designed and synthesized 75 analogues of hit 1 with the aim of increasing the affinity of this chemotype toward pre-miR21. Biochemical activity was evaluated using the fluorescence assay developed in Dr. Duca’s laboratory, which guided the further evolution of this chemotype. During this process, a well-defined structure–activity relationship (SAR) was established, suggesting possible target engagement. Ligand–target interactions were further investigated during my research period at AstraZeneca (Sweden) through NMR structural studies. For achieving these outcomes, fluorinated probes were produced through solid-phase synthesis by systematically introducing them into the pre-miR21 structure. These studies provided insights into the RNA secondary structure and demonstrated that the synthesized compounds establish specific interactions with pre-miR21. Biochemical target engagement was also assessed using an NMR-based assay, providing orthogonal validation to the fluorescence-based method. SAR analysis established across both approaches showed consistent trends, thus confirming the putative modulation of miR21 by small molecule compounds featuring this novel chemotype. Having confirmed favourable biochemical ligand–target interactions, the cellular activity of the most promising novel compounds was evaluated by assessing modulation of both mature miR21 levels and related targets. The data produced demonstrated that the identified compounds are indeed active in PANC-1 cell-line and exhibit preliminary indications of possible target engagement. Overall, throughout this activity set-up, fragment-based drug discovery was shown to be effective from initial screening to hit evolution, yielding small molecules that remain amenable to further optimization and that may serve as starting points for a broader medicinal chemistry campaign.
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