Discovery of Carbohydrate Sulfotransferase Inhibitors from a Kinase-Directed Library

Armstrong, Joshua I.; Portley, Adam R.; Chang, Young‐Tae; Nierengarten, David M.; Cook, Brian N.; Bowman, Kendra G.; Bishop, Anthony C.; Gray, Nathanael S.; Shokat, Kevan M.; Schultz, Peter G.; Bertozzi, Carolyn R.

doi:10.1002/(sici)1521-3757(20000403)112:7<1359::aid-ange1359>3.0.co;2-d

Cited by 23 publications

(22 citation statements)

References 20 publications

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“…Regulation of prokaryote-eukaryote interactions is known to involve sulfurylation of small molecules (35), and sulfotransferases play a significant role in modulating normal and pathogenic biological processes (26,38,39). ASST from the periplasm of uropathogenic E. coli strains is up-regulated in the uropathogenic habitat (10) and may therefore be involved in host-pathogen interactions.…”

Section: Discussionmentioning

confidence: 99%

A structural and biochemical basis for PAPS-independent sulfuryl transfer by aryl sulfotransferase from uropathogenic Escherichia coli

Malojcic

Owen

Grimshaw

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Sulfotransferases are a versatile class of enzymes involved in numerous physiological processes. In mammals, adenosine 3-phosphate-5-phosphosulfate (PAPS) is the universal sulfuryl donor, and PAPS-dependent sulfurylation of small molecules, including hormones, sugars, and antibiotics, is a critical step in hepatic detoxification and extracellular signaling. In contrast, little is known about sulfotransferases in bacteria, which make use of sulfurylated molecules as mediators of cell-cell interactions and host-pathogen interactions. Bacterial arylsulfate sulfotransferases (also termed aryl sulfotransferases), in contrast to PAPS-dependent sulfotransferases, transfer sulfuryl groups exclusively among phenolic compounds in a PAPS-independent manner. Here, we report the crystal structure of the virulence factor arylsulfate sulfotransferase (ASST) from the prototypic, pyelonephritogenic Escherichia coli strain CFT073 at 2.0-Å resolution, and 2 catalytic intermediates, at 2.1-Å and 2.4-Å resolution, with substrates bound in the active site. ASST is one of the largest periplasmic enzymes and its 3D structure differs fundamentally from all other structurally characterized sulfotransferases. Each 63.8-kDa subunit of the ASST homodimer comprises a 6-bladed ␤-propeller domain and a C-terminal ␤-sandwich domain. The active sites of the dimer are situated at the center of the channel formed by each ␤-propeller and are defined by the side chains of His-252, His-356, Arg-374, and His-436. We show that ASST follows a ping-pong bi-bi reaction mechanism, in which the catalytic residue His-436 undergoes transient sulfurylation, a previously unreported covalent protein modification. The data provide a framework for understanding PAPS-independent sulfotransfer and a basis for drug design targeting this bacterial virulence factor. beta propeller ͉ crystal structure ͉ pyelonephritis ͉ uropathogenic Escherichia coli CFT073 periplasm S ulfotransferases catalyze the transfer of a sulfuryl group from an activated donor to an acceptor and are essential for numerous physiological processes, such as sulfur metabolism, liver detoxification, signal transduction, hormone regulation, viral entry, and molecular recognition. A variety of small molecules, including hormones, sugars, and antibiotics, have been shown to undergo 3Ј-phosphate-5Ј-phosphosulfate (PAPS)-dependent sulfurylation (1, 2), and sulfotransferases are recognized as modulators of prokaryote-eukaryote interactions (3).In mammals, sulfoconjugation and glucuronidation represent the dominant mechanisms for detoxification of endogenous and exogenous compounds bearing phenolic groups (4, 5). Therefore, hepatic sulfotransferases are of considerable toxicological and pharmacological interest. In addition to mammalian sulfotransferases, arylsulfate sulfotransferases of commensal intestinal bacteria have been proposed to play a role in the detoxification of phenolic compounds (6, 7). Although a number of eukaryotic sulfotransferases have been extensively studied, much less is known about ba...

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Section: Discussionmentioning

confidence: 99%

A structural and biochemical basis for PAPS-independent sulfuryl transfer by aryl sulfotransferase from uropathogenic Escherichia coli

Malojcic

Owen

Grimshaw

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…We confirmed by DSF that TPST ligands act as competitive active-site inhibitors of peptide sulphation, creating a new impetus to develop novel screening approaches to discover TPST inhibitors. Standard biochemical assays often involve the detection of 35 S-based substrate sulphation derived from 35 S-labelled PAPS, and require enzymatic co-factor synthesis and time-consuming radioactive solid-phase chromatography (typically HPLC) procedures [80, 81]. In contrast, our peptide sulphation assay detects modification in real-time using a simple mobility shift assay, which is quantified by comparing the ratio of the sulphated and non-sulphated fluorescent substrates.…”

Section: Discussionmentioning

confidence: 99%

New tools for evaluating protein tyrosine sulphation: Tyrosyl Protein Sulphotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors

Eyers

Byrne

Ngamlert

et al. 2018

Preprint

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7Protein tyrosine sulphation is a post-translational modification (PTM) best known for regulating 1 8 extracellular protein-protein interactions. Tyrosine sulphation is catalysed by two Golgi-resident 1 9 enzymes termed Tyrosyl Protein Sulpho Transferases (TPSTs) 1 and 2, which transfer sulphate from 2 0 the co-factor PAPS (3'-phosphoadenosine 5'-phosphosulphate) to a context-dependent tyrosine in a 2 1 protein substrate. A lack of quantitative tyrosine sulphation assays has hampered the development of 2 2 chemical biology approaches for the identification of small molecule inhibitors of tyrosine sulphation. 3In this paper, we describe the development of a non-radioactive mobility-based enzymatic assay for 2 4 TPST1 and TPST2, through which the tyrosine sulphation of synthetic fluorescent peptides can be 2 5 rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of 2 6 TPST1 and 2 to different classes of small molecules, including the anti-angiogenic compound suramin 2 7 and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set (PKIS), we 2 8 identified oxindole-based inhibitors of the Ser/Thr kinase RAF as low micromolar inhibitors of 2 9 TPST1/2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor 3 0RAF265, were also TPST inhibitors in vitro. We propose that target-validated protein kinase 3 1 inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the 3 2 sulphotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine 3 3 sulphation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST 3 4 ligands and RAF protein kinase inhibitors. 3 5

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“…These compounds lack activity in cells, most likely due to their inability to access the 2-O-sulfotransferase in the Golgi of intact cells. Bertozzi and coworkers screened purine derivatives and found compounds with high selectivity towards individual sulfotransferases, suggesting that subtle differences in the PAPS binding sites can be exploited (Armstrong et al, 2000;Kehoe et al, 2002;. Inhibitors of HA or KS synthesis have not been described.…”

Section: Enzyme Inhibitorsmentioning

confidence: 99%

Glycan Antagonists and Inhibitors: A Fount for Drug Discovery

Brown¹,

Crawford²

2007

Critical Reviews in Biochemistry and Molecular Biology

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Glycans, the carbohydrate chains of glycoproteins, proteoglycans, and glycolipids, represent a relatively unexploited area for drug development compared with other macromolecules. This review describes the major classes of glycans synthesized by animal cells, their mode of assembly, and available inhibitors for blocking their biosynthesis and function. Many of these agents have proven useful for studying the biological activities of glycans in isolated cells, during embryological development, and in physiology. Some are being used to develop drugs for treating metabolic disorders, cancer, and infection, suggesting that glycans are excellent targets for future drug development.

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Discovery of Carbohydrate Sulfotransferase Inhibitors from a Kinase-Directed Library

Cited by 23 publications

References 20 publications

A structural and biochemical basis for PAPS-independent sulfuryl transfer by aryl sulfotransferase from uropathogenic Escherichia coli

A structural and biochemical basis for PAPS-independent sulfuryl transfer by aryl sulfotransferase from uropathogenic Escherichia coli

New tools for evaluating protein tyrosine sulphation: Tyrosyl Protein Sulphotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors

Glycan Antagonists and Inhibitors: A Fount for Drug Discovery

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