Fluorescent screening of glyco-active compounds with GlycoFluo technology

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Vers un nouveau traitement des mucopolysaccharidoses ? Recherche d’inhibiteurs de la β1,4-galactosyltransférase 7 (β4GalT7) par des approches combinées de criblage expérimental et virtuel

International audienc

Vers un nouveau traitement des mucopolysaccharidoses ? Recherche d’inhibiteurs de la β1,4-galactosyltransférase 7 (β4GalT7) par des approches combinées de criblage expérimental et virtuel

International audienc

Chemical synthesis and development of modified xylosides as potential inhibitors targeting β4GalT7, a key enzyme in glycosaminoglycan biosynthesis initiation.

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Synthesis of a library of variously modified 4-methylumbelliferyl xylosides and a structure–activity study of human β4GalT7

International audienceProteoglycans (PGs) are complex macromolecules that are composed of glycosaminoglycan (GAG)chains covalently attached to a core protein through a tetrasaccharide linker. The biosynthesis of PGs iscomplex and involves a large number of glycosyltranferases. Here we present a structure–activity study ofhuman β4GalT7, which transfers the first Gal residue onto a xyloside moiety of the linkage region. Anefficient and regiocontrolled synthesis of a library of modified analogs of 4-methylumbelliferyl xyloside(XylMU) is reported herein. Hydroxyl groups at the position C-2, C-3 or C-4 have been epimerized and/orreplaced by a hydrogen or a fluorine, while the anomeric oxygen was replaced by either a sulfur or asulfone. The effect of these compounds on human β4GalT7 activity in vitro and on GAG biosynthesisin cellulo was then evaluated

A versatile strategy to synthesize N -methyl-anthranilic acid-labelled glycoprobes for fluorescence-based screening assays

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Probing the Acceptor Active Site Organization of the Human Recombinant beta 1,4-Galactosyltransferase 7 and Design of Xyloside-based Inhibitors

International audienceBackground: Glycosyltransferase inhibitors have important applications in therapeutics and as chemical biology tools. Results: The human 1,4-galactosyltransferase 7 enzyme active site was mapped by modeling, mutagenesis, and in vitro/in cellulo assays, and novel inhibitors were synthesized. Conclusion: An efficient inhibitor of 1,4-galactosyltransferase 7 and glycosaminoglycan synthesis was obtained. Significance: This inhibitory molecule can be exploited to investigate glycosaminoglycan biology and modulate glycosaminoglycan synthesis in therapeutics. Among glycosaminoglycan (GAG) biosynthetic enzymes, the human 1,4-galactosyltransferase 7 (h4GalT7) is characterized by its unique capacity to take over xyloside derivatives linked to a hydrophobic aglycone as substrates and/or inhibitors. This glycosyltransferase is thus a prime target for the development of regulators of GAG synthesis in therapeutics. Here, we report the structure-guided design of h4GalT7 inhibitors. By combining molecular modeling, in vitro mutagenesis, and kinetic measurements, and in cellulo analysis of GAG anabolism and decorin glycosylation, we mapped the organization of the acceptor binding pocket, in complex with 4-methylumbelliferone-xylopyranoside as prototype substrate. We show that its organization is governed, on one side, by three tyrosine residues, Tyr(194), Tyr(196), and Tyr(199), which create a hydrophobic environment and provide stacking interactions with both xylopyranoside and aglycone rings. On the opposite side, a hydrogen-bond network is established between the charged amino acids Asp(228), Asp(229), and Arg(226), and the hydroxyl groups of xylose. We identified two key structural features, i.e. the strategic position of Tyr(194) forming stacking interactions with the aglycone, and the hydrogen bond between the His(195) nitrogen backbone and the carbonyl group of the coumarinyl molecule to develop a tight binder of h4GalT7. This led to the synthesis of 4-deoxy-4-fluoroxylose linked to 4-methylumbelliferone that inhibited h4GalT7 activity in vitro with a K-i 10 times lower than the K-m value and efficiently impaired GAG synthesis in a cell assay. This study provides a valuable probe for the investigation of GAG biology and opens avenues toward the development of bioactive compounds to correct GAG synthesis disorders implicated in different types of malignancies