Small molecule splicing modulators that disrupt O-GlcNAc homeostasis

O-Linked N-acetylglucosamine (O-GlcNAc) is a nucleocytoplasmic post-translational modification that is tightly regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Dysregulation of O-GlcNAc in human disease has motivated efforts to therapeutically modulate O-GlcNAc. Drug repurposing efforts can accelerate these campaigns and unveil how clinically relevant compounds and pathways intersect with O-GlcNAc. Here we report the results of three parallel drug repurposing screens against the O-GlcNAc cycling enzymes in cells and in vitro that reveal kinase inhibitors GSK690693 and Y-33075 act as splicing modulators that disrupt O-GlcNAc homeostasis and simultaneously downregulate OGT and OGA. GSK690693 inhibits intron processing and Y-33075 alters exon inclusion. These effects on splicing are independent of their respective annotated targets, AKT and ROCK, and are distinct from OGT and OGA inhibitors and similar kinase inhibitors. Evaluation of a panel of splicing modulators revealed three more potent compounds (OTS964, indisulam, GNF2133) that similarly downregulate OGT and OGA through distinct splicing profiles. These findings reveal new chemotypes of splicing modulators and strategies to disrupt O-GlcNAc homeostasis.