title: Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro.
abstract: Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[3H]glucosamine, [35S]sulfate, and L-[14C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha-(3) and beta-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[3H]glucosamine, but not of [35S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[14C]leucine incorporation into total protein synthesis. These results suggest that analogs 3-5 exhibit an inhibitory effect on D-[3H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[3H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-alpha-(6) and beta-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[3H]glucosamine and [35S]sulfate incorporation into isolated GAGs, namely to approximately 7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (approximately 40 kDa) than for control cultures (approximately 77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D-[3H]glucosamine and [35S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.
authors.affiliation: Department of Chemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
authors.forename: Ali
authors.initials: A
authors.lastname: Berkin
authors.forename: Walter A
authors.initials: WA
authors.lastname: Szarek
authors.forename: Robert
authors.initials: R
authors.lastname: Kisilevsky
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D005260:Female; D005944:Glucosamine / Q000378:metabolism; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D022781:Hepatocytes / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D051379:Mice; D014176:Protein Biosynthesis / Q000187:drug effects*; D013431:Sulfates / Q000378:metabolism
chemical_list: D006025:Glycosaminoglycans; D013431:Sulfates; D007930:Leucine; D005944:Glucosamine; D000117:Acetylglucosamine