Potent inhibitors of the human RNA ligase Rlig1 highlights its role in RNA integrity maintenance under oxidative cellular stress.

Human RNA ligase 1 (Rlig1) catalyzes the ligation of 5'-phosphate to 3'-hydroxyl ends a conserved three-step mechanism. Rlig1-deficient HEK293 cells exhibit reduced cell viability and RNA integrity under oxidative stress, suggesting Rlig1's role in RNA repair maintenance. Reactive oxygen species (ROS) are linked to various diseases, including neurodegenerative disorders and cancer, where RNA damage has significant effects. This study identifies and characterizes Rlig1 inhibitors to elucidate its role in RNA metabolism. We developed a fluorescence resonance energy transfer (FRET)-based assay to monitor RNA ligation and screened a library of 13 026 bioactive small molecules. SGI-1027 emerged as a promising lead compound, and structure-activity relationship (SAR) studies revealed that the terminal residues play a key role in its inhibitory effect. In total 22 SGI-1027 derivatives were synthesized and tested, providing insights into the structural requirements for effective Rlig1 inhibition. Three derivatives showed low micromolar IC values and minimal cytotoxicity in HEK293 cells under physiological conditions. The combination of Rlig1 inhibition and oxidative stress led to reduced cell viability and compromised RNA integrity, reinforcing Rlig1's role in RNA maintenance. These findings provide a foundation for developing novel therapeutics aimed at targeting RNA maintenance pathways in conditions of dysregulated ROS levels.