Targeted Protein O-GlcNAcylation Using Bifunctional Small Molecules

ABSTRACT Protein O-linked β- N -acetylglucosamine modification (O-GlcNAcylation) plays a crucial role in regulating essential cellular processes. The disruption of O-GlcNAcylation homeostasis has been linked to various human diseases, including cancer, diabetes, and neurodegeneration. However, there are limited chemical tools for protein- and site-specific O-GlcNAc modification, rendering the precise study of O-GlcNAcylation challenging. To address this, we have developed first-in-class heterobifunctional small molecules, named O-GlcNAcylation targeting chimeras (OGTACs), which enable protein-specific O-GlcNAcylation in cells. OGTACs promote O-GlcNAcylation of proteins such as BRD4, CK2α, and EZH2 in cellulo by recruiting O-GlcNAc transferase (OGT), with temporal and magnitude control. Mass spectrometry data revealed that OGTACs induced site-selective O-GlcNAcylation of BRD4. Overall, OGTACs represent a promising approach for inducing protein-specific O-GlcNAcylation, thus enabling functional dissection and offering new directions for O-GlcNAc-targeting therapeutic development.