Discovery And Mechanistic Studies Of Small Molecule Inhibitors Targeting The O-GlcNAc Protein OGT

As the most abundant post-translational modification,glycosylation affects the function of a wide range of proteins and lipids.O-Glc NAcylation,a unique glycosylation modification on nuclear and cytoplasmic proteins,is relevant to a number of physiological processes such as cellular transcription and protein translation.OGT,as an enzyme that adds O-Glc NAc to target proteins,is aberrantly expressed in a variety of diseases including cancer,diabetes,and neurodegenerative diseases.Current small-molecule inhibitors of OGT are relatively homogeneous,have low selectivity and inhibit OGT activity mainly by binding to the donor substrate UDP-Glc NAc pocket,which process may affect other intracellular glycosylation modifications.Therefore,the search for small molecule inhibitors targeting OGT with new binding modes has become the target of this study.This research work is divided into three main areas:I.Establishment of a high-throughput screening method for HTRF.By constructing,expressing and purifying OGT,a high-throughput screening system for HTRF based on the enzymatic activity function of OGT was established,and the stability and feasibility of the system were tested.II.Screening and corroboration of OGT small molecule inhibitors.The HTRF high-throughput screening system was used to screen 1966 compounds and identified the seedling compound CDDO with IC₅₀=6.56±1.69μM.¹H-NMR and MST demonstrated that CDDO bound directly to OGT with K_d=1.7μM.HDX-MS demonstrated that CDDO bound in the pocket formed by the TPR domain and the N-terminal structural domain of OGT.HDX-MS results were validated by enzyme activity competition experiments,which demonstrated that changes in the concentration of OSMI-4 did not affect the IC₅₀ value of CDDO.An Alpha Screen method was developed to demonstrate the inhibitory activity of CDDO analogues against OGT,and finally molecular docking was used to predict the binding pattern of CDDO and its analogues.Molecular docking predicted that the change in activity of CDDO and its structural analogues was due to the formation of hydrogen bonds with Leu492 and Asn526 at different amounts and distances.III.Structural resolution of OGT.The predicted results of molecular docking were verified by culturing the co-crystallized protein of OGT-CDDO.The crystal quality was improved by improving the purification conditions of OGT,the pooling conditions of the apo crystalline protein and the Soaking time,and finally the apo crystals of OGT protein with a resolution of 3.8?were obtained.Subsequently,the crystal culture conditions will continue to be optimized to analyze the specific binding mode of CDDO and OGT.In the present work,small molecule inhibitors targeting OGT were discovered and validated by high-throughput screening.While most of the existing OGT inhibitors bind in the catalytic region,CDDO was found for the first time to bind in the TPR domain and in the pocket of the N-terminal structural domain.both CDDO and its analogues inhibit OGT to varying degrees and have good potential for modification.In conclusion,this topic has developed the first high-throughput screening method for HTRF based on the enzymatic activity function of OGT.A small molecule inhibitor CDDO with a novel binding site was discovered and confirmed.Finally,computer-aided drug design was applied to predict the binding mode of CDDO,which laid a good foundation for the subsequent discovery and modification of OGT inhibitors.