Abstract 3154: Targeting of the Elongator tRNA-modifier complex through high-throughput screening revealed series of compounds with novel mechanism of action

Numerous human diseases, such as cancers and neurodegenerative disorders, have been associated with the dysregulation of tRNA-modifying enzymes’ activity. These post-transcriptional modifications can affect tRNA structure stability, codon-anticodon base pairing during ribosomal decoding, translation efficiency, and proper protein folding during protein synthesis, impacting both cell cycle and metabolism. It is therefore not surprising that several tRNA-modifying enzymes have recently emerged as attractive therapeutic targets in drug discovery. Nevertheless, these enzymes often form large complexes with multiple subunits, and assessing their activity in vitro using reconstituted protein complexes and tRNA substrates is particularly challenging.The aim of the present study was to identify inhibitors of a cancer-implicated tRNA-modifying enzyme. For this purpose, we have developed a TR-FRET-based protein-RNA interaction assay suitable for high-throughput screening (HTS) in 384 MTP format. The protein-RNA interaction was assessed using a tagged recombinant catalytic subunit and a biotinylated RNA oligo derived from a specific tRNA. The optimization through buffer component screening and the validation with tool inhibitors yielded a thorough biochemical characterization of the in vitro protein-RNA binding. A HTS campaign with a proprietary library of ∼100,000 compounds was performed in search of protein-RNA interaction inhibitors. A secondary assay was used to identify those hit compounds interfering with the primary assay's readout system. The primary screening hit rate was 1 % (cut-off: 49% inhibition) and 84% of those hits were confirmed as putative protein-RNA interaction inhibitors with IC50 values in the nM-µM range and no interference effects.A few of these primary hits have progressed to the hit-to-lead stage, and the preliminary results obtained thus far are highly promising.This study illustrates a hit discovery program targeting the interaction between the catalytic subunit of a tRNA-modifying enzyme and a tRNA-derived RNA, aimed at identifying specific inhibitors. Targeting tRNA modification systems represents an attractive strategy for treating human cancers. Citation Format: Mariantonietta Rubino, Kerstin-Maike Schmitz, Andrea Lauri, Debora Zian, Nazareno Menegatti, Samuel Bradberry, Francesca Rapino, Pierre Close, Daniele Carettoni, Cinzia Nucci, Angela Molteni, Jean-Francois Rolland. Targeting of the Elongator tRNA-modifier complex through high-throughput screening revealed series of compounds with novel mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3154.