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Inhibitors incorporating zinc-binding groups target the GlcNAc-PI de-N-acetylase in Trypanosoma brucei, the causative agent of African sleeping sickness.

Abdelwahab NZ, Crossman AT, Sullivan L, Ferguson MA, Urbaniak MD - Chem Biol Drug Des (2012)

Bottom Line: We recently reported the synthesis of eight deoxy-2-C-branched monosaccharides containing carboxylic acid, hydroxamic acid, or N-hydroxyurea substituents at the C2 position that may act as zinc-binding groups.Here, we describe the synthesis of a glucocyclitol-phospholipid incorporating a hydroxamic acid moiety and report the biochemical evaluation of the monosaccharides and the glucocyclitol-phospholipid as inhibitors of the trypanosome deNAc in the cell-free system and against recombinant enzyme.Monosaccharides with carboxylic acid or hydroxamic acid substituents were found to be the inhibitors of the trypanosome deNAc with IC(50) values 0.1-1.5mM and the glucocyclitol-phospholipid was found to be a dual inhibitor of the deNAc and the α1-4-mannose transferase with an apparent IC(50)= 19±0.5μm.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK.

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Inhibition of Trypanosoma brucei glycosylphosphatidylinositol (GPI) biosynthesis in the cell-free system. Compounds 11–19 (10 mm) were incubated with the T. brucei cell-free system for 5 min prior to priming with GlcNAc-PI and GDP-[3H]Man to stimulate the production of radiolabelled mannosylated GPI intermediates. Glycolipid products were extracted, separated by high-performance thin-layer chromatography, and visualized by fluorography. DPM – dolichol-phosphate-mannose, M1 – Man1GlcN-PI, M2 – Man2GlcN-PI, M3 – Man3GlcN-PI, aM3 – Man3GlcN-(acyl)PI, A’– EtNPMan3GlcN-PI. PI, phosphatidylinositol.
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fig04: Inhibition of Trypanosoma brucei glycosylphosphatidylinositol (GPI) biosynthesis in the cell-free system. Compounds 11–19 (10 mm) were incubated with the T. brucei cell-free system for 5 min prior to priming with GlcNAc-PI and GDP-[3H]Man to stimulate the production of radiolabelled mannosylated GPI intermediates. Glycolipid products were extracted, separated by high-performance thin-layer chromatography, and visualized by fluorography. DPM – dolichol-phosphate-mannose, M1 – Man1GlcN-PI, M2 – Man2GlcN-PI, M3 – Man3GlcN-PI, aM3 – Man3GlcN-(acyl)PI, A’– EtNPMan3GlcN-PI. PI, phosphatidylinositol.

Mentions: The ability of the compounds 11–19 to inhibit the deNAc was initially assessed in vitro using the T. brucei cell-free system (cfs), i.e., washed trypanosome membranes that are competent in GPI biosynthesis. Because de-N-acetylation of GlcNAc-PI must precede the addition of the three mannose residues (13), the activity of the deNAc can be indirectly monitored by measuring production of mannosylated GPI biosynthetic intermediates. In the assay, the cfs is primed with GDP-[3H]Man and synthetic GlcNAc-PI 3 with and without inhibitor, and the radiolabelled mannosylated products separated by hptlc, quantified radiometrically, and visualized by fluorography. The compounds 11–19 were tested at an initial concentration of 10 mm in the cell-free system (Figure 4). Compounds 13, 15, 17, and 19 produced <10% inhibition of the formation of radiolabelled mannosylated products compared to the DMSO control, while 11, 12, 15, 16, and 18 all produced >80% inhibition. The potency of the latter compounds was then determined using eight-point potency curves in triplicate (Table 1). Notably, the α/β anomers 17 and 18 gave >100-fold difference in potency. These compounds contain a thiophenyl group, predicted to adopt an axial position in the α-anomer 17, and an equatorial position in the β-anomer 18, which could potentially lead to differences in the abilities of the two anomers to fit into the active site of the enzyme. This result is surprising, given that it has previously been observed that the deNAc is able to de-N-acetylate both the natural substrate GlcNAc-α-PI 1 and the unnatural GlcNAc-β-PI (17).


Inhibitors incorporating zinc-binding groups target the GlcNAc-PI de-N-acetylase in Trypanosoma brucei, the causative agent of African sleeping sickness.

Abdelwahab NZ, Crossman AT, Sullivan L, Ferguson MA, Urbaniak MD - Chem Biol Drug Des (2012)

Inhibition of Trypanosoma brucei glycosylphosphatidylinositol (GPI) biosynthesis in the cell-free system. Compounds 11–19 (10 mm) were incubated with the T. brucei cell-free system for 5 min prior to priming with GlcNAc-PI and GDP-[3H]Man to stimulate the production of radiolabelled mannosylated GPI intermediates. Glycolipid products were extracted, separated by high-performance thin-layer chromatography, and visualized by fluorography. DPM – dolichol-phosphate-mannose, M1 – Man1GlcN-PI, M2 – Man2GlcN-PI, M3 – Man3GlcN-PI, aM3 – Man3GlcN-(acyl)PI, A’– EtNPMan3GlcN-PI. PI, phosphatidylinositol.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473218&req=5

fig04: Inhibition of Trypanosoma brucei glycosylphosphatidylinositol (GPI) biosynthesis in the cell-free system. Compounds 11–19 (10 mm) were incubated with the T. brucei cell-free system for 5 min prior to priming with GlcNAc-PI and GDP-[3H]Man to stimulate the production of radiolabelled mannosylated GPI intermediates. Glycolipid products were extracted, separated by high-performance thin-layer chromatography, and visualized by fluorography. DPM – dolichol-phosphate-mannose, M1 – Man1GlcN-PI, M2 – Man2GlcN-PI, M3 – Man3GlcN-PI, aM3 – Man3GlcN-(acyl)PI, A’– EtNPMan3GlcN-PI. PI, phosphatidylinositol.
Mentions: The ability of the compounds 11–19 to inhibit the deNAc was initially assessed in vitro using the T. brucei cell-free system (cfs), i.e., washed trypanosome membranes that are competent in GPI biosynthesis. Because de-N-acetylation of GlcNAc-PI must precede the addition of the three mannose residues (13), the activity of the deNAc can be indirectly monitored by measuring production of mannosylated GPI biosynthetic intermediates. In the assay, the cfs is primed with GDP-[3H]Man and synthetic GlcNAc-PI 3 with and without inhibitor, and the radiolabelled mannosylated products separated by hptlc, quantified radiometrically, and visualized by fluorography. The compounds 11–19 were tested at an initial concentration of 10 mm in the cell-free system (Figure 4). Compounds 13, 15, 17, and 19 produced <10% inhibition of the formation of radiolabelled mannosylated products compared to the DMSO control, while 11, 12, 15, 16, and 18 all produced >80% inhibition. The potency of the latter compounds was then determined using eight-point potency curves in triplicate (Table 1). Notably, the α/β anomers 17 and 18 gave >100-fold difference in potency. These compounds contain a thiophenyl group, predicted to adopt an axial position in the α-anomer 17, and an equatorial position in the β-anomer 18, which could potentially lead to differences in the abilities of the two anomers to fit into the active site of the enzyme. This result is surprising, given that it has previously been observed that the deNAc is able to de-N-acetylate both the natural substrate GlcNAc-α-PI 1 and the unnatural GlcNAc-β-PI (17).

Bottom Line: We recently reported the synthesis of eight deoxy-2-C-branched monosaccharides containing carboxylic acid, hydroxamic acid, or N-hydroxyurea substituents at the C2 position that may act as zinc-binding groups.Here, we describe the synthesis of a glucocyclitol-phospholipid incorporating a hydroxamic acid moiety and report the biochemical evaluation of the monosaccharides and the glucocyclitol-phospholipid as inhibitors of the trypanosome deNAc in the cell-free system and against recombinant enzyme.Monosaccharides with carboxylic acid or hydroxamic acid substituents were found to be the inhibitors of the trypanosome deNAc with IC(50) values 0.1-1.5mM and the glucocyclitol-phospholipid was found to be a dual inhibitor of the deNAc and the α1-4-mannose transferase with an apparent IC(50)= 19±0.5μm.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK.

Show MeSH
Related in: MedlinePlus