O-GlcNAc Transferase and Its Inhibitors

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O-GlcNAc transferase (OGT), one of essential mammalian enzymes, catalyses the transfer of N-acetylglucos- amine from UDP-N-acetylglucosamine (UDP-GlcNAc) to hydroxyl groups of serines and threonines (Ser/Thr) in proteins. O-GlcNAcylation is widely located in cytoplasm and nucleus. This kind of protein post-translational modification is involved in the regulation of many cellular signaling pathways and closely associated with the occurrences and developments of nu- merous critical illnesses. In animals, OGT is encoded by a single highly conserved gene. However, human OGT includes three different isoforms at least: nucleocytoplasmic OGT (ncOGT), mitochondrial OGT (mOGT) and short OGT (sOGT). All of the isoforms comprise two distinct regions: a multidomain catalytic region and an N-terminal region with different num- bers of tetratricopeptide repeats (TPRs), which is closely related to their subcellular localizations. The structures of the cata- lytic region and TPRs were reported and it was supported that OGT uses an ordered sequential bi-bi mechanism though the details of catalytic mechanism were not completely understood. To discover the inhibitors of OGT, several OGT activity assay methods were developed. The conventional method uses a radiolabeled glycosyl donor substrate such as UDP-( 3 H)-GlcNAc or UDP-( 14 C)-GlcNAc so that it is not suitable for in vivo test or rapid analysis. O-GlcNAc anti- body-based western blot was performed to measure the activity of OGT in vivo, but it is infeasible to determine low abun- dance protein with single O-GlcNAc. Three high throughput activity assays were reported: ligand displacement OGT assay, protease-protection assay strategy and azido-enzyme-linked immunosorbent assay (azido-ELISA). The first two methods have especially been used to find new OGT inhibitors through screening compound libraries, but the results have to be veri- fied by the conventional method. To date, OGT inhibitor activity of several candidates was evaluated and as found, two of them {2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-5-thio-α-D-glucopyranose and 4-methoxyphenyl 6-acetyl-2-oxobenzo(d)- oxazole-3(2H)-carboxylate} worked effectively in vivo and were valuable for understanding the functional roles of OGT. Nevertheless, there are still important questions to be answered in the research of OGT: (1) What is the general base that catalyzes O-GlcNAcylation reaction? (2) How can OGT recognize so many protein substances with the preservation of sub- stance specificity? (3) How can we develop robust high-throughput OGT assays and isoform-specific OGT inhibitors? In conclusion, there is a long way to go for comprehensive understanding of OGT.