Overview of the Assays to Probe O-Linked β-N-Acetylglucosamine Transferase Binding and Activity

Abstract

O-GlcNAcylation is a posttranslational modification that occurs at serine and threonine residues of protein substrates by the addition of O-linked β-d-N-acetylglucosamine (GlcNAc) moiety. Two enzymes are involved in this modification: O-GlcNac transferase (OGT), which attaches the GlcNAc residue to the protein substrate, and O-GlcNAcase (OGA), which removes it. This biological balance is important for many biological processes, such as protein expression, cell apoptosis, and regulation of enzyme activity. The extent of this modification has sparked interest in the medical community to explore OGA and OGT as therapeutic targets, particularly in degenerative diseases. While some OGA inhibitors are already in phase 1 clinical trials for the treatment of Alzheimer’s disease, OGT inhibitors still have a long way to go. Due to complex expression and instability, the discovery of potent OGT inhibitors is challenging. Over the years, the field has grappled with this problem, and scientists have developed a number of techniques and assays. In this review, we aim to highlight assays and techniques for OGT inhibitor discovery, evaluate their strength for the field, and give us direction for future bioassay methods.

Discovery of a New Drug-like Series of OGT Inhibitors by Virtual Screening

“…After carefully inspecting the predicted binding poses, we selected one to three hits from each cluster based on their synthetic accessibility and chemical diversity. Eighteen molecules were purchased from different vendors ( Table S2 ) and screened in vitro for OGT inhibition at 100 µM using a fluorescence-based transferase activity assay [ 32 , 33 ] ( Table 1 , Figure S1 ). Although six compounds were found to be active at this concentration, we identified Vs-5, Vs-51, and Vs-83 as the most potent and promising hits and therefore proceeded to measure their IC 50 values.…”

Can O-GIcNAc Transferase (OGT) Complex Be Used as a Target for the Treatment of Hematological Malignancies?

“…Expression of MLL-AF9 fusion genes has been associated with extramedullary tumor infiltration, high recurrence rates, and low survival [ 97 ]. An example is P2X7 (P2X7 belongs to the P2X family of purinergic receptors and is aberrantly expressed in various types of malignant tumors, including leukemia) [ 98 ]. MOF is a histone 4 lysine 16 (H4K16) acetyltransferase and is a member of the lysine acetyltransferase MYST family, which is named after its founding members, MOZ, YBF2, SAS2, and TIP60, with MOF being a member of the MYST family [ 31 ].…”

O-GlcNAcylation: The Underestimated Emerging Regulators of Skeletal Muscle Physiology

“…There are abundant phosphorylation and ubiquitination sites in the domains of glycoside hydrolase and the HAT-like domain [ 7 ], but the effect of these modifications at corresponding sites on OGA activity remains to be further determined. The advanced structure of OGA and the sites modified by various PTMs are shown in Figure 3 F. The O -GlcNAcylation of OGA at Ser 405 is located in the central highly disordered region, suggesting a role in the regulation of OGA-OGT interactions because this is the binding region of OGA-OGT [ 122 ]. OGA is also SUMOylated at Lys 358 and acetylated at Lys 599 , respectively [ 7 , 123 ].…”