The correlation between cellular O-GlcNAcylation and sensitivity to O-GlcNAc inhibitor in colorectal cancer cells.

The upregulation of O-GlcNAc signaling has long been implicated in the development and progression of numerous human malignancies, including colorectal cancer. In this study, we characterized eight colorectal cancer cell lines and one non-cancerous cell line for O-GlcNAc-related profiles such as the expression of OGT, OGA, and total protein O-GlcNAcylation, along with their sensitivity toward OSMI-1 (Os), an OGT inhibitor (OGTi). Indeed, Os dose-dependently suppressed the viability of all colorectal cancer cell lines tested. Among the three O-GlcNAc profiles, our results revealed that Os IC50 exhibited the strongest correlation with total protein O-GlcNAcylation (Pearson Correlation Coefficient r = -0.73), suggesting that total O-GlcNAcylation likely serves as a better predictive marker for OGTi sensitivity than OGT expression levels. Furthermore, we demonstrated that Os exhibited a synergistic relationship with regorafenib (Re). We believed that this synergism could be explained, at least in part, by the observed Re-mediated increase of cellular O-GlcNAcylation, which was counteracted by Os. Finally, we showed that the Os:Re combination suppressed the growth of NCI-H508 tumor spheroids. Overall, our findings highlighted OGTi as a potential anticancer agent that could be used in combination with other molecules to enhance the efficacy while minimizing adverse effects, and identified total cellular O-GlcNAcylation as a potential predictive marker for OGTi sensitivity.

O-GlcNAcylation inhibition redirects the response of colon cancer cells to chemotherapy from senescence to apoptosis.

The potential use of pro-senescence therapies, known as TIS (Therapy-Induced Senescence), for the treatment of colorectal cancer (CRC) generated significant interest since they require lower doses compared to those required for inducing apoptosis. However, the senescent cell cycle-arrested cancer cells are long-lived, and studies have revealed escape mechanisms contributing to tumor recurrence. To deepen our understanding of the survival pathways used by senescent cancer cells, we delved into the potential involvement of the hexosamine biosynthetic pathway (HBP). HBP provides UDP-GlcNAc, the substrate for O-GlcNAc transferase (OGT), which catalyzes O-GlcNAcylation, a post-translational modification implicated in regulating numerous cellular functions and aberrantly elevated in CRC. In this study, we demonstrated, in the p53-proficient colon cancer cell lines HCT116 and LS174T, that TIS induced by low-dose SN38 or etoposide treatment was accompanied with a decrease of GFAT (the rate limiting enzyme of the HBP), OGT and O-GlcNAcase (OGA) expression correlated with a slight reduction in O-GlcNAcylation levels. Further decreasing this level of O-GlcNAcylation by knocking-down GFAT or OGT redirected the cellular response to subtoxic chemotherapy doses from senescence to apoptosis, in correlation with an enhancement of DNA damages. Pharmacological inhibition of OGT with OSMI-4 in HCT116 and LS174T cells and in a patient-derived colon tumoroid model supported these findings. Taken together, these results suggest that combing O-GlcNAcylation inhibitors to low doses of conventional chemotherapeutic drugs could potentially reduce treatment side effects while preserving efficacy. Furthermore, this approach may increase treatment specificity, as CRC cells exhibit higher O-GlcNAcylation levels compared to normal tissues.

Inhibition of O-GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway.

The O-GlcNAc transferase (OGT) is an essential enzyme that mediates protein O-GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O-GlcNAcylation levels in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent mice by repressing the cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT (Ogt-/-) caused a marked reduction in tumor growth in both syngeneic mice tumor models and a genetic mice colorectal cancer (CRC) model induced by mutation of the Apc gene (Apcmin). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of the type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas or Sting from Ogt-/- cancer cells restored tumor growth, and this correlated with impaired CD8+ T-cell-mediated antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production in tumors. In summary, our results identify OGT-mediated genomic stability and activate cGAS-STING pathway as an important tumor-cell-intrinsic mechanism to repress antitumor immunity.

Identification of a Polypeptide Inhibitor of O-GlcNAc Transferase with Picomolar Affinity.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that binds thousands of different proteins, including substrates that it glycosylates and nonsubstrate interactors that regulate its biology. OGT also has one proteolytic substrate, the transcriptional coregulator host cell factor 1 (HCF-1), which it cleaves in a process initiated by glutamate side chain glycosylation at a series of central repeats. Although HCF-1 is OGT's most prominent binding partner, its affinity for the enzyme has not been quantified. Here, we report a time-resolved Förster resonance energy transfer assay to measure ligand binding to OGT and show that an HCF-1-derived polypeptide (HCF3R) binds with picomolar affinity to the enzyme (KD ≤ 85 pM). This high affinity is driven in large part by conserved asparagines in OGT's tetratricopeptide repeat domain, which form bidentate contacts to the HCF-1 peptide backbone; replacing any one of these asparagines with alanine reduces binding by more than 5 orders of magnitude. Because the HCF-1 polypeptide binds so tightly to OGT, we tested its ability to inhibit enzymatic function. We found that HCF3R potently inhibits OGT both in vitro and in cells and used this finding to develop a genetically encoded, inducible OGT inhibitor that can be degraded with a small molecule, allowing for reversible and tunable inhibition of OGT.

O-GlcNAcylation of MITF regulates its activity and CDK4/6 inhibitor resistance in breast cancer.

Cyclin-dependent kinases 4 and 6 (CDK4/6) play a pivotal role in cell cycle and cancer development. Targeting CDK4/6 has demonstrated promising effects against breast cancer. However, resistance to CDK4/6 inhibitors (CDK4/6i), such as palbociclib, remains a substantial challenge in clinical settings. Using high-throughput combinatorial drug screening and genomic sequencing, we find that the microphthalmia-associated transcription factor (MITF) is activated via O-GlcNAcylation by O-GlcNAc transferase (OGT) in palbociclib-resistant breast cancer cells and tumors. Mechanistically, O-GlcNAcylation of MITF at Serine 49 enhances its interaction with importin α/ß, thus promoting its translocation to nuclei, where it suppresses palbociclib-induced senescence. Inhibition of MITF or its O-GlcNAcylation re-sensitizes resistant cells to palbociclib. Moreover, clinical studies confirm the activation of MITF in tumors from patients who are palbociclib-resistant or undergoing palbociclib treatment. Collectively, our studies shed light on the mechanism regulating palbociclib resistance and present clinical evidence for developing therapeutic approaches to treat CDK4/6i-resistant breast cancer patients.

O-GlcNAc Modification Is a Promising Therapeutic Target for Diabetic Retinopathy.

Diabetic retinopathy (DR) is a very serious diabetes complication. Changes in the O-linked N-acetylglucosamine (O-GlcNAc) modification are associated with many diseases. However, its role in DR is not fully understood. In this research, we explored the effect of O-GlcNAc modification regulation by activating AMP-activated protein kinase (AMPK) in DR, providing some evidence for clinical DR treatment in the future. Bioinformatics was used to make predictions from the database, which were validated using the serum samples of diabetic patients. As an in vivo model, diabetic mice were induced using streptozotocin (STZ) injection with/without an AMPK agonist (metformin) or an AMPK inhibitor (compound C) treatment. Electroretinogram (ERG) and H&E staining were used to evaluate the retinal functional and morphological changes. In vitro, 661 w cells were exposed to high-glucose conditions, with or without metformin treatment. Apoptosis was evaluated using TUNEL staining. The protein expression was detected using Western blot and immunofluorescence staining. The angiogenesis ability was detected using a tube formation assay. The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in the serum changed in the DR patients in the clinic. In the diabetic mice, the ERG wave amplitude and retinal thickness decreased. In vitro, the apoptotic cell percentage and Bax expression were increased, and Bcl2 expression was decreased in the 661 w cells under high-glucose conditions. The O-GlcNAc modification was increased in DR. In addition, the expression of GFAT/TXNIP O-GlcNAc was also increased in the 661 w cells after the high-glucose treatment. Additionally, the Co-immunoprecipitation(CO-IP) results show that TXNIP interacted with the O-GlcNAc modification. However, AMPK activation ameliorated this effect. We also found that silencing the AMPKα1 subunit reversed this process. In addition, the conditioned medium of the 661 w cells may have affected the tube formation in vitro. Taken together, O-GlcNAc modification was increased in DR with photoreceptor cell degeneration and neovascularization; however, it was reversed after activating AMPK. The underlying mechanism is linked to the GFAT/TXNIP-O-GlcNAc modification signaling axis. Therefore, the AMPKα1 subunit plays a vital role in the process.

Discovery of two non-UDP-mimic inhibitors of O-GlcNAc transferase by screening a DNA-encoded library.

Finding potent inhibitors of O-GlcNAc transferase (OGT) has proven to be a challenge, especially because the diversity of published inhibitors is low. The large majority of available OGT inhibitors are uridine-based or uridine-like compounds that mimic the main interactions of glycosyl donor UDP-GlcNAc with the enzyme. Until recently, screening of DNA-encoded libraries for discovering hits against protein targets was dedicated to a few laboratories around the world, but has become accessible to wider public with the recent launch of the DELopen platform. Here we report the results and follow-up of a DNA-encoded library screening by using the DELopen platform. This led to the discovery of two new hits with structural features not resembling UDP. Small focused libraries bearing those two scaffolds were made, leading to low micromolar inhibition of OGT and elucidation of their structure-activity relationship.

Understanding and exploiting the roles of O-GlcNAc in neurodegenerative diseases.

O-GlcNAc is a common modification found on nuclear and cytoplasmic proteins. Determining the catalytic mechanism of the enzyme O-GlcNAcase (OGA), which removes O-GlcNAc from proteins, enabled the creation of potent and selective inhibitors of this regulatory enzyme. Such inhibitors have served as important tools in helping to uncover the cellular and organismal physiological roles of this modification. In addition, OGA inhibitors have been important for defining the augmentation of O-GlcNAc as a promising disease-modifying approach to combat several neurodegenerative diseases including both Alzheimer's disease and Parkinson's disease. These studies have led to development and optimization of OGA inhibitors for clinical application. These compounds have been shown to be well tolerated in early clinical studies and are steadily advancing into the clinic. Despite these advances, the mechanisms by which O-GlcNAc protects against these various types of neurodegeneration are a topic of continuing interest since improved insight may enable the creation of more targeted strategies to modulate O-GlcNAc for therapeutic benefit. Relevant pathways on which O-GlcNAc has been found to exert beneficial effects include autophagy, necroptosis, and processing of the amyloid precursor protein. More recently, the development and application of chemical methods enabling the synthesis of homogenous proteins have clarified the biochemical effects of O-GlcNAc on protein aggregation and uncovered new roles for O-GlcNAc in heat shock response. Here, we discuss the features of O-GlcNAc in neurodegenerative diseases, the application of inhibitors to identify the roles of this modification, and the biochemical effects of O-GlcNAc on proteins and pathways associated with neurodegeneration.

Discovery of 12 (BMS-986172) as a Highly Potent MGAT2 Inhibitor that Achieved Targeted Efficacious Exposures at a Low Human Dose for the Treatment of Metabolic Disorders.

A series of dihydropyridinone (DHP) compounds was prepared and evaluated for MGAT2 activity. The efforts led to the identification of novel tetrazolones with potent MGAT2 inhibitory activity and favorable in vitro profiles. Further tests of select analogues in mouse models revealed significant reduction in food intake and body weight. Subsequent studies in MGAT2 knockout mice with the lead candidate 12 (BMS-986172) showed on-target- and mechanism-based pharmacology. Moreover, its favorable pharmacokinetic (PK) profile and the lack of species variability in the glucuronidation potential resulted in a greater confidence level in the projection of a low dose for achieving targeted efficacious exposures in humans. Consistent with these projections, PK data from a phase 1 trial confirmed that targeted efficacious exposures could be achieved at a low dose in humans, which supported compound 12 as our second and potentially superior development candidate for the treatment of various metabolic disorders.

O-GlcNAc transferase regulates p21 protein levels and cell proliferation through the FoxM1-Skp2 axis in a p53-independent manner.

The protein product of the CDKN1A gene, p21, has been extensively characterized as a negative regulator of the cell cycle. Nevertheless, it is clear that p21 has manifold complex and context-dependent roles that can be either tumor suppressive or oncogenic. Most well studied as a transcriptional target of the p53 tumor suppressor protein, there are other means by which p21 levels can be regulated. In this study, we show that pharmacological inhibition or siRNA-mediated reduction of O-GlcNAc transferase (OGT), the enzyme responsible for glycosylation of intracellular proteins, increases expression of p21 in both p53-dependent and p53-independent manners in nontransformed and cancer cells. In cells harboring WT p53, we demonstrate that inhibition of OGT leads to p53-mediated transactivation of CDKN1A, while in cells that do not express p53, inhibiting OGT leads to increased p21 protein stabilization. p21 is normally degraded by the ubiquitin-proteasome system following ubiquitination by, among others, the E3 ligase Skp-Cullin-F-box complex; however, in this case, we show that blocking OGT causes impairment of the Skp-Cullin-F-box ubiquitin complex as a result of disruption of the FoxM1 transcription factor-mediated induction of Skp2 expression. In either setting, we conclude that p21 levels induced by OGT inhibition correlate with cell cycle arrest and decreased cancer cell proliferation.

Inhibition of O-GlcNAc Transferase Alters the Differentiation and Maturation Process of Human Monocyte Derived Dendritic Cells.

The O-GlcNAcylation is a posttranslational modification of proteins regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase. These enzymes regulate the development, proliferation and function of cells, including the immune cells. Herein, we focused on the role of O-GlcNAcylation in human monocyte derived dendritic cells (moDCs). Our study suggests that inhibition of OGT modulates AKT and MEK/ERK pathways in moDCs. Changes were also observed in the expression levels of relevant surface markers, where reduced expression of CD80 and DC-SIGN, and increased expression of CD14, CD86 and HLA-DR occurred. We also noticed decreased IL-10 and increased IL-6 production, along with diminished endocytotic capacity of the cells, indicating that inhibition of O-GlcNAcylation hampers the transition of monocytes into immature DCs. Furthermore, the inhibition of OGT altered the maturation process of immature moDCs, since a CD14medDC-SIGNlowHLA-DRmedCD80lowCD86high profile was noticed when OGT inhibitor, OSMI-1, was present. To evaluate DCs ability to influence T cell differentiation and polarization, we co-cultured these cells. Surprisingly, the observed phenotypic changes of mature moDCs generated in the presence of OSMI-1 led to an increased proliferation of allogeneic T cells, while their polarization was not affected. Taken together, we confirm that shifting the O-GlcNAcylation status due to OGT inhibition alters the differentiation and function of moDCs in in vitro conditions.

Screening Hit to Clinical Candidate: Discovery of BMS-963272, a Potent, Selective MGAT2 Inhibitor for the Treatment of Metabolic Disorders.

MGAT2 inhibition is a potential therapeutic approach for the treatment of metabolic disorders. High-throughput screening of the BMS internal compound collection identified the aryl dihydropyridinone compound 1 (hMGAT2 IC50 = 175 nM) as a hit. Compound 1 had moderate potency against human MGAT2, was inactive vs mouse MGAT2 and had poor microsomal metabolic stability. A novel chemistry route was developed to synthesize aryl dihydropyridinone analogs to explore structure-activity relationship around this hit, leading to the discovery of potent and selective MGAT2 inhibitors 21f, 21s, and 28e that are stable to liver microsomal metabolism. After triaging out 21f due to its inferior in vivo potency, pharmacokinetics, and structure-based liabilities and tetrazole 28e due to its inferior channel liability profile, 21s (BMS-963272) was selected as the clinical candidate following demonstration of on-target weight loss efficacy in the diet-induced obese mouse model and an acceptable safety and tolerability profile in multiple preclinical species.

OSMI-1 Enhances TRAIL-Induced Apoptosis through ER Stress and NF-κB Signaling in Colon Cancer Cells.

Levels of O-GlcNAc transferase (OGT) and hyper-O-GlcNAcylation expression levels are associated with cancer pathogenesis. This study aimed to find conditions that maximize the therapeutic effect of cancer and minimize tissue damage by combining an OGT inhibitor (OSMI-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We found that OSMI-1 treatment in HCT116 human colon cancer cells has a potent synergistic effect on TRAIL-induced apoptosis signaling. Interestingly, OSMI-1 significantly increased TRAIL-mediated apoptosis by increasing the expression of the cell surface receptor DR5. ROS-induced endoplasmic reticulum (ER) stress by OSMI-1 not only upregulated CHOP-DR5 signaling but also activated Jun-N-terminal kinase (JNK), resulting in a decrease in Bcl2 and the release of cytochrome c from mitochondria. TRAIL induced the activation of NF-κB and played a role in resistance as an antiapoptotic factor. During this process, O-GlcNAcylation of IκB kinase (IKK) and IκBα degradation occurred, followed by translocation of p65 into the nucleus. However, combination treatment with OSMI-1 counteracted the effect of TRAIL-mediated NF-κB signaling, resulting in a more synergistic effect on apoptosis. Therefore, the combined treatment of OSMI-1 and TRAIL synergistically increased TRAIL-induced apoptosis through caspase-8 activation. Conclusively, OSMI-1 potentially sensitizes TRAIL-induced cell death in HCT116 cells through the blockade of NF-κB signaling and activation of apoptosis through ER stress response.

Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity.

[Figure: see text].

Recent Advances about the Applications of Click Reaction in Chemical Proteomics.

Despite significant advances in biological and analytical approaches, a comprehensive portrait of the proteome and its dynamic interactions and modifications remains a challenging goal. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to elucidate protein composition, distribution, and relevant physiological and pharmacological functions. Click chemistry focuses on the development of new combinatorial chemical methods for carbon heteroatom bond (C-X-C) synthesis, which have been utilized extensively in the field of chemical proteomics. Click reactions have various advantages including high yield, harmless by-products, and simple reaction conditions, upon which the molecular diversity can be easily and effectively obtained. This paper reviews the application of click chemistry in proteomics from four aspects: (1) activity-based protein profiling, (2) enzyme-inhibitors screening, (3) protein labeling and modifications, and (4) hybrid monolithic column in proteomic analysis.

Elimination of GPI2 suppresses glycosylphosphatidylinositol GlcNAc transferase activity and alters GPI glycan modification in Trypanosoma brucei.

Many eukaryotic cell-surface proteins are post-translationally modified by a glycosylphosphatidylinositol (GPI) moiety that anchors them to the cell membrane. The biosynthesis of GPI anchors is initiated in the endoplasmic reticulum by transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol. This reaction is catalyzed by GPI GlcNAc transferase, a multisubunit complex comprising the catalytic subunit Gpi3/PIG-A as well as at least five other subunits, including the hydrophobic protein Gpi2, which is essential for the activity of the complex in yeast and mammals, but the function of which is not known. To investigate the role of Gpi2, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote and important model organism that initially provided the first insights into GPI structure and biosynthesis. We generated insect-stage (procyclic) trypanosomes that lack TbGPI2 and found that in TbGPI2-null parasites, (i) GPI GlcNAc transferase activity is reduced, but not lost, in contrast with yeast and human cells, (ii) the GPI GlcNAc transferase complex persists, but its architecture is affected, with loss of at least the TbGPI1 subunit, and (iii) the GPI anchors of procyclins, the major surface proteins, are underglycosylated when compared with their WT counterparts, indicating the importance of TbGPI2 for reactions that occur in the Golgi apparatus. Immunofluorescence microscopy localized TbGPI2 not only to the endoplasmic reticulum but also to the Golgi apparatus, suggesting that in addition to its expected function as a subunit of the GPI GlcNAc transferase complex, TbGPI2 may have an enigmatic noncanonical role in Golgi-localized GPI anchor modification in trypanosomes.

Development of tricyclic N-benzyl-4-hydroxybutanamide derivatives as inhibitors of GABA transporters mGAT1-4 with anticonvulsant, antinociceptive, and antidepressant activity.

γ-Aminobutyric acid (GABA) neurotransmission has a significant impact on the proper functioning of the central nervous system. Numerous studies have indicated that inhibitors of the GABA transporters mGAT1-4 offer a promising strategy for the treatment of several neurological disorders, including epilepsy, neuropathic pain, and depression. Following our previous results, herein, we report the synthesis, biological evaluation, and structure-activity relationship studies supported by molecular docking and molecular dynamics of a new series of N-benzyl-4-hydroxybutanamide derivatives regarding their inhibitory potency toward mGAT1-4. This study allowed us to identify compound 23a (N-benzyl-4-hydroxybutanamide bearing a dibenzocycloheptatriene moiety), a nonselective GAT inhibitor with a slight preference toward mGAT4 (pIC50 = 5.02 ± 0.11), and compound 24e (4-hydroxy-N-[(4-methylphenyl)-methyl]butanamide bearing a dibenzocycloheptadiene moiety) with relatively high inhibitory activity toward mGAT2 (pIC50 = 5.34 ± 0.09). In a set of in vivo experiments, compound 24e successively showed predominant anticonvulsant activity and antinociception in the formalin model of tonic pain. In contrast, compound 23a showed significant antidepressant-like properties in mice. These results were consistent with the available literature data, which indicates that, apart from seizure control, GABAergic neurotransmission is also involved in the pathophysiology of several psychiatric diseases, however alternative mechanisms underlying this action cannot be excluded. Finally, it is worth noting that the selected compounds showed unimpaired locomotor skills that have been indicated to give reliable results in behavioral assays.

Novel bisubstrate uridine-peptide analogues bearing a pyrophosphate bioisostere as inhibitors of human O-GlcNAc transferase.

Protein O-linked ß-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), an essential post-translational as well as cotranslational modification, is the attachment of ß-D-N-acetylglucosamine to serine and threonine residues of nucleocytoplasmic proteins. An aberrant O-GlcNAc profile on certain proteins has been implicated in metabolic diseases such as diabetes and cancer. Inhibitors of O-GlcNAc transferase (OGT) are valuable tools to study the cell biology of protein O-GlcNAc modification. In this study we report novel uridine-peptide conjugate molecules composed of an acceptor peptide covalently linked to a catalytically inactive donor substrate analogue that bears a pyrophosphate bioisostere and explore their inhibitory activities against OGT by a radioactive hOGT assay. Further, we investigate the structural basis of their activities via molecular modelling, explaining their lack of potency towards OGT inhibition.

Emerging roles of protein O-GlcNAcylation in cardiovascular diseases: Insights and novel therapeutic targets.

Cardiovascular diseases (CVDs) are the leading cause of death globally. While the major focus of pharmacological and non-pharmacological interventions has been on targeting disease pathophysiology and limiting predisposing factors, our understanding of the cellular and molecular mechanisms underlying the pathogenesis of CVDs remains incomplete. One mechanism that has recently emerged is protein O-GlcNAcylation. This is a dynamic, site-specific reversible post-translational modification of serine and threonine residues on target proteins and is controlled by two enzymes: O-linked ß-N-acetylglucosamine transferase (OGT) and O-linked ß-N-acetylglucosaminidase (OGA). Protein O-GlcNAcylation alters the cellular functions of these target proteins which play vital roles in pathways that modulate vascular homeostasis and cardiac function. Through this review, we aim to give insights on the role of protein O-GlcNAcylation in cardiovascular diseases and identify potential therapeutic targets in this pathway for development of more effective medicines to improve patient outcomes.

Bisubstrate Ether-Linked Uridine-Peptide Conjugates as O-GlcNAc Transferase Inhibitors.

The O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a master regulator of installing O-GlcNAc onto serine or threonine residues on a multitude of target proteins. Numerous nuclear and cytosolic proteins of varying functional classes, including translational factors, transcription factors, signaling proteins, and kinases are OGT substrates. Aberrant O-GlcNAcylation of proteins is implicated in signaling in metabolic diseases such as diabetes and cancer. Selective and potent OGT inhibitors are valuable tools to study the role of OGT in modulating a wide range of effects on cellular functions. We report linear bisubstrate ether-linked uridine-peptide conjugates as OGT inhibitors with micromolar affinity. In vitro evaluation of the compounds revealed the importance of donor substrate, linker and acceptor substrate in the rational design of bisubstrate analogue inhibitors. Molecular dynamics simulations shed light on the binding of this novel class of inhibitors and rationalized the effect of amino acid truncation of acceptor peptide on OGT inhibition.