Screening And Identification Of An O-linked N-acetylglucosamine Transferase Inhibitor

O-linked-N-acetylglucosamine?O-GlcNAc?modification is a protein post-translational modification?PTM?,which is widely present in mammalian cytoplasm and nucleus.Unlike complex glycan modifications on the cell membrane,O-GlcNAc modifications are reversible monosaccharide modifications that occur on the serine/threonine residues of intracellular soluble proteins.O-GlcNAc modification is similar to protein phosphorylation modification and is a dynamically changing process.This process refers to the glycosyl donor UDP-GlcNAc linked to the bare serine/threonine residue of the protein under the catalytic action of O-linked N-acetylglucosamine transferase?OGT?to complete the O-GlcNAc modification.Under the action of the corresponding glycosidase?OGA?,O-GlcNAc is hydrolyzed from the peptide chain of the protein to complete deglycosylation.Among the reported small-molecule inhibitors of O-GlcNAc,the natural product tetrahydroflavone?L01?developed earlier in this group showed lower IC₅₀ values and had the advantage of less cytotoxicity.On this basis,this study further screened its structural analogs to obtain 3,8'-Biapigenin with better OGT inhibitory activity.The molecular docking and binding energy analysis of 10 kinds of L01 natural product analogues and OGT were performed to compare the OGT inhibitory activities of these compounds in a cell-free reaction system.The comparative analysis showed that 3,8'-Biapigenin aglycone has an OGT inhibitory activity better than that of L01.Molecular dynamics simulation results show that 3,8'-Biapigenin can form stable hydrogen bond interaction with Phe 837?Gln 839?Ala 896?His 920?Thr922 near the OGT substrate binding pocket.Further,the intracellular activity of the molecule was detected,and Western blot method was used to find that 3,8'-Biapigenin can inhibit OGT activity in a time and concentration gradient manner,thereby reducing the O-GlcNAc modification of human cervical cancer Hela cell.Wound healing and Transwell methods were used to observe the effect of 3,8'-Biapigenin on the migration and invasion potential of Hela cells.The results showed that 3,8'-Biapigenin has better tumor cell metastasis inhibitory activity than L01.Through online prediction and exogenous expression and other means,this paper proves that the topoisomerase???TOP2A?protein in the cell has O-GlcNAc modification,and uses 3,8'-Biapigenin to treat 293T cells that overexpress TOP2A.It was found to have O-GlcNAc modification that inhibits TOP2A.The research results in this paper prove that 3,8'-Biapigenin has the effect of modifying the extracellular and intracellular inhibitory protein O-GlcNAc,thereby preventing the migration and invasion of tumor cells.These results can provide a reference for the study of O-GlcNAc small molecule inhibitors and O-GlcNAc modification functions,and provide relevant tools for the treatment of tumor metastasis related research.