title: An efficient approach to the discovery of potent inhibitors against glycosyltransferases.
abstract: We describe a standardized approach for searching potent and selective inhibitors of glycosyltransferases by high throughput quantitative MALDI-TOFMS-based screening of focused compound libraries constructed by 1,3-dipolar cycloaddition of the desired azidosugar nucleotides with various alkynes. An aminooxy-functionalized reagent with a stable isotope was conjugated with oligosaccharides to afford glycopeptides as acceptor substrates with improved ion sensitivity. Enhanced ionization potency of new substrates allowed for MALDI-TOFMS-based facile and quantitative analysis of enzymatic glycosylation in the presence of glycosyl donor substrates. A non-natural synthetic sugar nucleotide was identified to be the first highly specific inhibitor for rat recombinant alpha2,3-(N)-sialyltransferase (alpha2,3ST, IC(50) = 8.2 microM), while this compound was proved to become a favorable substrate for rat recombinant alpha2,6-(N)-sialyltransferase (alpha2,6ST, K(m) = 125 microM). Versatility of this strategy was demonstrated by identification of two selective inhibitors for human recombinant alpha1,3-fucosyltransferase V (alpha1,3-FucT, K(i) = 293 nM) and alpha1,6-fucosyltransferase VIII (alpha1,6-FucT, K(i) = 13.8 microM).
authors.affiliation: Graduate School of Life Science and Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Kita-ku, Sapporo, Japan.
authors.forename: Kensaku
authors.initials: K
authors.lastname: Hosoguchi
authors.forename: Takahiro
authors.initials: T
authors.lastname: Maeda
authors.forename: Jun-Ichi
authors.initials: J
authors.lastname: Furukawa
authors.forename: Yasuro
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hinou
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Sekiguchi
authors.forename: Hiroko
authors.initials: H
authors.lastname: Togame
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takemoto
authors.forename: Hirosato
authors.initials: H
authors.lastname: Kondo
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D000480:Alkynes / Q000138:chemical synthesis* / Q000737:chemistry; D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000737:chemistry; D016208:Databases, Factual; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry; D006020:Glycopeptides / Q000138:chemical synthesis / Q000737:chemistry; D016695:Glycosyltransferases / Q000037:antagonists & inhibitors*; D006801:Humans; D009711:Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
chemical_list: D000480:Alkynes; D001386:Azides; D006020:Glycopeptides; D009711:Nucleotides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D016695:Glycosyltransferases; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; C020559:Glycoprotein 6-alpha-L-fucosyltransferase; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase