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O‐GlcNAc transferase (OGT) is the only enzyme that catalyzes the post‐translational modification of proteins at Ser/Thr with a single β‐N‐acetylglucosamine (O‐GlcNAcylation). Its activity has been associated with chronic diseases such as cancer, diabetes and neurodegenerative disease. Although numerous OGT substrates have been identified, its accepted substrate scope can still be refined. We report here an attempt to better define the peptide‐recognition requirements of the OGT active site by using mRNA display, taking advantage of its extremely high throughput to assess the substrate potential of a library of all possible nonamer peptides. An antibody‐based selection process is described here that is able to enrich an OGT substrate peptide from such a library, but with poor absolute recovery. Following four rounds of selection for O‐GlcNAcylated peptides, sequencing revealed 14 peptides containing Ser/Thr, but these were shown by luminescence‐coupled assays and peptide microarray not to be OGT substrates. By contrast, subsequent testing of an N‐terminal tag approach showed exemplary recovery. Our approach demonstrates the power of genetically encoded libraries for selection of peptide substrates, even from a very low initial starting abundance and under suboptimal conditions, and emphasizes the need to consider the binding biases of antibodies and both C‐ and N‐terminal tags in profiling peptide substrates by high‐throughput display., Improving the odds: In vitro mRNA display‐based ultra‐high‐throughput selection is shown here to be a feasible approach to selectively enrich peptide substrates of O‐GlcNAc transferase from the background of a random library. We identify and solve two crucial challenges to high‐throughput profiling of peptide substrates of glycosyltransferases, working towards a universally applicable system.

O-GlcNAcylation is an essential post-translational modification installed by the enzyme O-β-N-acetyl-d-glucosaminyl transferase (OGT). Modulating this enzyme would be extremely valuable to better understand its role in the development of serious human pathologies, such as diabetes and cancer. However, the limited availability of potent and selective inhibitors hinders the validation of this potential therapeutic target. To explore new chemotypes that target the active site of OGT, we performed virtual screening of a large library of commercially available compounds with drug-like properties. We purchased samples of the most promising virtual hits and used enzyme assays to identify authentic leads. Structure-activity relationships of the best identified OGT inhibitor were explored by generating a small library of derivatives. Our best hit displays a novel uridine mimetic scaffold and inhibited the recombinant enzyme with an IC50 value of 7 µM. The current hit represents an excellent starting point for designing and developing a new set of OGT inhibitors that may prove useful for exploring the biology of OGT. Nasl. z nasl. zaslona. Opis vira z dne 21. 3. 2022. Št. članka: 1996. Bibliografija: str. 19-21. Abstract. ARRS European Union’s Horizon2020, projekt

O-GlcNAc transferase (OGT) is the only enzyme that catalyzes the post-translational modification of proteins at Ser/Thr with a single β-N-acetylglucosamine (O-GlcNAcylation). Its activity has been associated with chronic diseases such as cancer, diabetes and neurodegenerative disease. Although numerous OGT substrates have been identified, its accepted substrate scope can still be refined. We report here an attempt to better define the peptide-recognition requirements of the OGT active site by using mRNA display, taking advantage of its extremely high throughput to assess the substrate potential of a library of all possible nonamer peptides. An antibody-based selection process is described here that is able to enrich an OGT substrate peptide from such a library, but with poor absolute recovery. Following four rounds of selection for O-GlcNAcylated peptides, sequencing revealed 14 peptides containing Ser/Thr, but these were shown by luminescence-coupled assays and peptide microarray not to be OGT substrates. By contrast, subsequent testing of an N-terminal tag approach showed exemplary recovery. Our approach demonstrates the power of genetically encoded libraries for selection of peptide substrates, even from a very low initial starting abundance and under suboptimal conditions, and emphasizes the need to consider the binding biases of antibodies and both C- and N-terminal tags in profiling peptide substrates by high-throughput display.

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.