Structure-Based Evolution of Low Nanomolar O-GlcNAc Transferase Inhibitors

Martin, Sara; Tan, Zhi-Wei; Itkonen, Harri M.; Duveau, Damien Y.; Paulo, João A.; Janetzko, John; Boutz, Paul L.; Törk, Lisa; Moss, Frederick A.; Thomas, Craig J.; Gygi, Steven P.; Lazarus, Michael B.; Walker, Suzanne

doi:10.1021/jacs.8b07328

Cited by 135 publications

(178 citation statements)

References 25 publications

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“…MEF cells were grown in Dulbecco’s Modified Eagle Medium (Gibco, USA) supplemented with 10% FBS and 1X Penicillin-Streptomycin solution (Corning) at 37°C in 5% CO 2 . OSMI-2 was synthesized as previously described, cycloheximide (239765-1ML) and Thiamet-G (SML0244) was purchased from Sigma-Aldrich (19). For siRNA transfection, HEK293T cells were transfected with the corresponding siRNA following the manufacturer’s protocol.…”

Section: Methodsmentioning

confidence: 99%

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O-GlcNAc regulates gene expression by controlling detained intron splicing

Tan

Fei

Paulo

et al. 2020

Preprint

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ABSTRACTIntron detention in precursor RNAs serves to regulate expression of a substantial fraction of genes in eukaryotic genomes. How detained intron (DI) splicing is controlled is poorly understood. Here we show that a ubiquitous post-translational modification called O-GlcNAc, which is thought to integrate signaling pathways as nutrient conditions fluctuate, controls detained intron splicing. Using specific inhibitors of the enzyme that installs O-GlcNAc (O-GlcNAc transferase, or OGT) and the enzyme that removes O-GlcNAc (O-GlcNAcase, or OGA), we first show that O-GlcNAc regulates splicing of the highly conserved detained introns in OGT and OGA to control mRNA abundance in order to buffer O-GlcNAc changes. We show that OGT and OGA represent two distinct paradigms for how DI splicing can control gene expression. We also show that when DI splicing of the O-GlcNAc-cycling genes fails to restore O-GlcNAc homeostasis, there is a global change in detained intron levels. Strikingly, almost all detained introns are spliced more efficiently when O-GlcNAc levels are low, yet other alternative splicing pathways change minimally. Our results demonstrate that O-GlcNAc controls detained intron splicing to tune system-wide gene expression, providing a means to couple nutrient conditions to the cell’s transcriptional regime.

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Section: Methodsmentioning

confidence: 99%

“…We have developed a class of OGT inhibitors useful for interrogating acute cellular changes that occur when OGT function is disrupted (18,19). Here we probe system-wide effects of OGT inhibition at short time points.…”

Section: Introductionmentioning

confidence: 99%

O-GlcNAc regulates gene expression by controlling detained intron splicing

Tan

Fei

Paulo

et al. 2020

Preprint

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“…In the commercialised UDP-Glo assay (Promega), UDP release is coupled to generation of ATPa nd subsequently al uminescent signal. [23] The quinolinone-6-sulfonamide core in these applications acts as amimic of the sugar donorsnucleotide base,suggesting much broader applicability.Coupled assays can also be useful, such as using glycosyl transfer to block the activity of one or more exo-acting glycosidases on afluorescent substrate. [19] Afurther alternative uses release of one or two equivalents of inorganic phosphate from the nucleotides by ap hosphatase to allow its quantification using malachitebased reagents,a lthough this precludes the use of phosphate buffer.…”

Section: Searching For Inhibitorsmentioning

confidence: 99%

“…In an example application of this approach from the Walker group,i nitial hits against O-GlcNAc transferase from an expanded version of the the commercial ChemDiv library [21] were diversified through combinatorial chemistry based on ac onserved quinolinone-6-sulfonamide core, [22] before the elucidation of an X-ray crystal structure allowed structure-based optimisation to am olecule with low-nanomolar potency in vitro and al owmicromolar EC 50 in cellular assays ( Figure 2B). [23] The quinolinone-6-sulfonamide core in these applications acts as amimic of the sugar donorsnucleotide base,suggesting much broader applicability.Coupled assays can also be useful, such as using glycosyl transfer to block the activity of one or more exo-acting glycosidases on afluorescent substrate. [24] Peptide display on phage or mRNA [25] can be used to select peptides with as trong affinity for in principle any carbohydrate-active enzyme or carbohydrate-binding protein, thereby allowing discovery of peptide-based inhibitors.…”

Section: Searching For Inhibitorsmentioning

confidence: 99%

High‐Throughput Approaches in Carbohydrate‐Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors, Evolution, and Discovery

Chao

Jongkees

2019

Angew Chem Int Ed

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Carbohydrates are attached and removed in living systems through the action of carbohydrate‐active enzymes such as glycosyl transferases and glycoside hydrolases. The molecules resulting from these enzymes have many important roles in organisms, such as cellular communication, structural support, and energy metabolism. In general, each carbohydrate transformation requires a separate catalyst, and so these enzyme families are extremely diverse. To make this diversity manageable, high‐throughput approaches look at many enzymes at once. Similarly, high‐throughput approaches can be a powerful way of finding inhibitors that can be used to tune the reactivity of these enzymes, either in an industrial, a laboratory, or a medicinal setting. In this review, we provide an overview of how these enzymes and inhibitors can be sought using techniques such as high‐throughput natural product and combinatorial library screening, phage and mRNA display of (glyco)peptides, fluorescence‐activated cell sorting, and metagenomics.

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“…However, the O-GlcNAc modification, like many cellular PTMs, cannot be tuned on particular glycoproteins within the cell. Global alteration of O-GlcNAc levels can be achieved through manipulating gene expression or chemical inhibitors (7,8), but relating biological effects to a specific glycoprotein requires extensive follow-up studies. Elimination of the O-GlcNAc modification on specific target proteins is possible following glycosite mapping and mutagenic removal of the glycosite from the protein in cells.…”

Section: Introductionmentioning

confidence: 99%

Selective protein O-GlcNAcylation in cells by a proximity-directed O-GlcNAc transferase

Ramirez

Aonbangkhen

et al. 2019

Preprint

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O-Linked N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Yet, the study of post-translational modifications like O-GlcNAc has been limited by the lack of strategies to induce O-GlcNAcylation on a target protein in cells. Here, we report a generalizable genetic strategy to induce O-GlcNAc to specific target proteins in cells using a nanobody as a proximitydirecting agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT(4), a truncated form of OGT, yielded a nanobody-OGT(4) construct that selectively delivered O-GlcNAc to the target protein (e.g., JunB, cJun, Nup62) and reduced alteration of global O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the selective increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein. Finally, we demonstrate the ability to selectively target endogenous ⍺-synuclein for glycosylation in HEK293T cells. Thus, the use of nanobodies to redirect OGT substrate selection is a versatile strategy to induce glycosylation of desired target proteins in cells that will facilitate discovery of O-GlcNAc functions and provide a mechanism to engineer O-GlcNAc signaling. The proximity-directed OGT approach for protein-selective O-GlcNAcylation is readily translated to additional protein targets and nanobodies that may constitute a generalizable strategy to control post-translational modifications in cells. Significance StatementNature uses post-translational modifications (PTMs) like glycosylation as a mechanism to alter protein signaling and function. However, the study of these modified proteins in cells is confined to loss-of-function strategies, such as mutagenic elimination of the modification site. Here, we report a generalizable strategy for induction of O-GlcNAc to a protein target in cells. The O-GlcNAc modification is installed by O-GlcNAc transferase (OGT) to thousands of nucleocytoplasmic proteins. Fusion of a nanobody to OGT enables the selective increase of O-GlcNAc levels on a series of target proteins. The described approach will facilitate direct studies of O-GlcNAc and its regulatory enzymes and drive new approaches to engineer protein signaling via a strategy that may be conceptually translatable to additional PTMs.

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Structure-Based Evolution of Low Nanomolar O-GlcNAc Transferase Inhibitors

Cited by 135 publications

References 25 publications

O-GlcNAc regulates gene expression by controlling detained intron splicing

O-GlcNAc regulates gene expression by controlling detained intron splicing

High‐Throughput Approaches in Carbohydrate‐Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors, Evolution, and Discovery

Selective protein O-GlcNAcylation in cells by a proximity-directed O-GlcNAc transferase

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