title: Probing the substrate specificity of Trypanosoma brucei GlcNAc-PI de-N-acetylase with synthetic substrate analogues.
abstract: A series of synthetic analogues of 1-D-(2-amino-2-deoxy-α-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate), consisting of 7 variants of either the D-myo-inositol, D-GlcpN or the phospholipid components, were prepared and tested as substrates and inhibitors of GlcNAc-PI de-N-acetylase, a genetically validated drug target enzyme responsible for the second step in the glycosylphosphatidylinositol (GPI) biosynthetic pathway of Trypanosoma brucei. The D-myo-inositol in the physiological substrate was successfully replaced by cyclohexanediol and is still a substrate for T. brucei GlcNAc-PI de-N-acetylase. However, this compound became sensitive to the stereochemistry of the glycoside linkage (the β-anomer was neither substrate or inhibitor) and the structure of the lipid moiety (the hexadecyl derivatives were inhibitors). Chemistry was successfully developed to replace the phosphate with a sulphonamide, but the compound was neither a substrate or an inhibitor, confirming the importance of the phosphate for molecular recognition. We also replaced the glucosamine by an acyclic analogue, but this also was inactive, both as a substrate and inhibitor. These findings add significantly to our understanding of substrate and inhibitor binding to the GlcNAc-PI de-N-acetylase enzyme and will have a bearing on the design of future inhibitors.
authors: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK. m.a.j.ferguson@dundee.ac.uk i.h.gilbert@dundee.ac.uk.
authors: Amy S
authors: AS
authors: Capes
authors: Arthur
authors: A
authors: Crossman
authors: Michael D
authors: MD
authors: Urbaniak
authors: Sophie H
authors: SH
authors: Gilbert
authors: Michael A J
authors: MA
authors: Ferguson
authors: Ian H
authors: IH
authors: Gilbert
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D008968:Molecular Conformation; D010716:Phosphatidylinositols / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; D010716:Phosphatidylinositols; D000581:Amidohydrolases; C087696:N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase; D005944:Glucosamine