title: Development of a simple homogeneous assay to screen for inhibitors of N-acetylglucosamine-6-sulfotransferases.
abstract: L-selectin, a leukocyte adhesion molecule, plays a central role in lymphocyte homing to secondary lymphoid tissue and to certain sites of inflammation. Carbohydrate sulfation was implicated in this process, when it was demonstrated that carbohydrate sulfotransferase-mediated sulfation of N-acetylglucosamine (GlcNAc) within sialyl Lewis X of cognate endothelial ligands for L-selectin was an essential modification for L-selectin binding. The recently identified GlcNAc-6-sulfotransferases GlcNAc6ST-1 and -2, which facilitate GlcNAc sulfation by catalyzing the transfer of a sulfonyl group from 3(')-phosphoadenosine 5(')-phosphosulfate (PAPS) to the 6-hydroxy group of the acceptor GlcNAc moiety, contribute to the biosynthesis of the 6-sulfosialyl Lewis X motif. Due to their pivotal role in L-selectin ligand biosynthesis, this enzyme class has recently emerged as an important and relatively unexplored class of potential targets for anti-inflammatory therapy. However, no inhibitors have been reported to date and screening for lead inhibitors has been hampered by the lack of simple assay formats suitable for high-throughput screening. Here, we report the development of a simple homogeneous in vitro sulfotransferase assay using a newly synthesized biotinylated glycoside as a substrate. The assay is based on GlcNAc6ST-2-mediated [35S]sulfate transfer from [35S]PAPS to the biotinylated glycoside and subsequent detection using streptavidin-coated SPA beads. K(m) values with partially purified GlcNAc6ST-2 for PAPS and the biotinylated glycoside were estimated to be 8.4 and 34.5 microM, respectively. The sulfotransferase reaction could be inhibited by 3('),5(')-ADP with an IC(50) of 2.1 microM. The assay can be operated in 384-well format; is characterized by a high signal-to-noise ratio, low variation, and excellent Z factors; and is highly suitable for high-throughput screening.
authors.affiliation: Bayer Yakuhin Ltd., Research Center Kyoto, 6-5-1-3 Kunimidai, Kizu-cho, Soraku-gun, Japan. hiroko.togame.ht@bayer.co.jp
authors.forename: Hiroko
authors.initials: H
authors.lastname: Togame
authors.forename: Makoto
authors.initials: M
authors.lastname: Shimazaki
authors.forename: Atsuko
authors.initials: A
authors.lastname: Yamato
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Watanabe
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Saito
authors.forename: Peter
authors.initials: P
authors.lastname: Reinemer
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D001331:Automation; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D002460:Cell Line; D004338:Drug Combinations; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006001:Glycoconjugates / Q000138:chemical synthesis / Q000378:metabolism; D006027:Glycosides / Q000138:chemical synthesis / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D013422:Sulfamonomethoxine / Q000037:antagonists & inhibitors; D015238:Sulfotransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013997:Time Factors; D014295:Trimethoprim / Q000037:antagonists & inhibitors; D000097811:Carbohydrate Sulfotransferases
chemical_list: D000244:Adenosine Diphosphate; D001710:Biotin; D004338:Drug Combinations; D004791:Enzyme Inhibitors; D006001:Glycoconjugates; D006027:Glycosides; D010724:Phosphoadenosine Phosphosulfate; D013422:Sulfamonomethoxine; D015238:Sulfotransferases; D014295:Trimethoprim; D000097811:Carbohydrate Sulfotransferases; C062251:sulfaton