Discovery of a novel OGT inhibitor through high-throughput screening based on Homogeneous Time-Resolved Fluorescence (HTRF)

O-GlcNAcylation is a specific type of post-translational glycosylation modification, which is regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Aberrant overexpression of OGT is associated with the development of many solid tumors. In this study, we have developed and optimized a sensitive Homogeneous Time-Resolved Fluorescence (HTRF) assay then identified a novel OGT inhibitor CDDO (also called Bardoxolone) through a high-throughput screening (HTS) based on HTRF assay. Further characterization suggested that CDDO is an effective OGT inhibitor with an IC50 value of 6.56 ± 1.69 μM. CPMG-NMR analysis confirmed that CDDO is a direct binder of OGT with a binding affinity (Kd) of approximately 1.7 μM determined by the MST analysis. Moreover, HDX-MS analysis indicated that CDDO binds to the TPR domain and N-Terminal domain of OGT, which was further confirmed by the enzymatic competition experiments as the binding of CDDO to OGT was not affected by the catalytic site binding inhibitor OSMI-4. Our docking modeling analysis further predicted the possible interactions between CDDO and OGT, providing informative molecular basis for further optimization of the inhibitor in the future. Together, our results suggested CDDO is a new inhibitor of OGT with a distinct binding pocket from the reported OGT inhibitors. Our work paved a new direction for developing OGT inhibitors driven by novel mechanisms.