Chemical biology tools for the O-GlcNAc modification: Determining systems-level functions and druggability.

How can a single monosaccharide control nearly every human cellular feature? This question has hounded the O-GlcNAc field since 1984. Despite identifying thousands of O-GlcNAc proteins, high-throughput datasets have only deepened the mystery. This Current Opinion highlights chemical biology tools (current as of 2023-2026) that reveal coordinated O-GlcNAc networks in physiology and disease. We review five areas: (1) systems-level maps of tissue-specific OGT interactomes and substrates; (2) spatiotemporal tools for precise glycosylation manipulation; (3) multiplexed detection assays for O-GlcNAc activities alongside other PTMs; (4) targeted modulation via nontraditional inhibitors, noncatalytic OGT scaffolding, and ligand-directed assembly; and (5) disease models uncovering tissue-specific effects. Recent OGA inhibitor clinical challenges in Phase 1 and 2 studies pose existential questions about drugging O-GlcNAc, but recent advances covered in this Opinion propose insights for safe therapeutic targeting. Through the lens of new chemical biology tools, we see detailed patterns in how nutrient-responsive O-GlcNAcylation subtly regulates cellular decision-making.