title: A fluorescence-based high-throughput assay to identify inhibitors of tyrosylprotein sulfotransferase activity.
abstract: Tyrosylprotein sulfotransferases (TPSTs) are Golgi-resident enzymes that catalyze the transfer of a sulfuryl group to the side chain hydroxyl of tyrosine residues. Sulfotyrosine residues are involved in protein-protein interactions in the extracellular space. These interactions are important for chemokines to bind cognate receptor, for cell adhesion and trafficking, and for pathogen entry into cells. To better understand the role of TPSTs in cellular processes and disease states, we are interested in identifying small molecules to modulate TPST activity in experimental systems. Towards that end, we developed a fluorescent peptide assay for TPST2 activity. Here, we demonstrate that this assay can be used to screen the 1280 compound LOPAC library in a 384-well format and in a high-throughput manner. We identified 19 primary hits for a hit rate of 1.5%. Three of the primary hits were verified by dose-response assay and confirmed as inhibitors by a secondary mass spectrometry assay for TPST activity. One hit, suramin, possessed inhibitory properties consistent with a competitive inhibitor of substrate binding and molecular docking revealed a good fit into the TPST2 substrate-binding pocket. This assay can be used to screen larger libraries to identify small molecules that inhibit TPST sulfotransferase activity.
authors.affiliation: Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware St. SE, 7-215 PWB, MMC 204, Minneapolis, MN 55455, USA.
authors.forename: Wenbo
authors.initials: W
authors.lastname: Zhou
authors.affiliation: Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware St. SE, 7-215 PWB, MMC 204, Minneapolis, MN 55455, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware St. SE, 7-215 PWB, MMC 204, Minneapolis, MN 55455, USA.
authors.forename: Jiashu
authors.initials: J
authors.lastname: Xie
authors.affiliation: Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware St. SE, 7-215 PWB, MMC 204, Minneapolis, MN 55455, USA. Electronic address: gerag012@umn.edu.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Geraghty
mesh_terms: D016367:Baculoviridae; D002853:Chromatography, Liquid; D020650:Combinatorial Chemistry Techniques; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D006801:Humans; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D062105:Molecular Docking Simulation; D010455:Peptides / Q000737:chemistry; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D013498:Suramin / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D007527:Isoenzymes; D010455:Peptides; D013498:Suramin; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase
keywords: Aurintricarboxylic acid (PubChem CID: 2259); Fluorescence; Fluphenazine dihydrochloride (PubChem CID: 67356); Guanidinyl-naltrindole di-trifluoroacetate (PubChem CID: 9853099); High-throughput; Sulfotransferase; Suramin; Suramin (sodium salt) (PubChem CID: 8514); TPST