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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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Inhibitors target the GlcNAc-PI de-N-acetylase. (A) Inhibition of the Trypanosoma brucei cell-free system by 11 and 18 (10 mm) when primed with either GlcNAc-PI or GlcN-PI. Conditions as Figure 4. (B) Inhibition of recombinant T. brucei GlcNAc-PI de-N-acetylase by 11 measured by electrospray tandem mass spectrometry. The intensity of the reaction product GlcN-IPC184 (m/z 672) is normalized to the turnover in uninhibited control. PI, phosphatidylinositol.
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fig05: Inhibitors target the GlcNAc-PI de-N-acetylase. (A) Inhibition of the Trypanosoma brucei cell-free system by 11 and 18 (10 mm) when primed with either GlcNAc-PI or GlcN-PI. Conditions as Figure 4. (B) Inhibition of recombinant T. brucei GlcNAc-PI de-N-acetylase by 11 measured by electrospray tandem mass spectrometry. The intensity of the reaction product GlcN-IPC184 (m/z 672) is normalized to the turnover in uninhibited control. PI, phosphatidylinositol.

Mentions: The indirect cfs assay is unable to distinguish between the inhibition of the deNAc and inhibition of the first mannosyltransferase (MT1), because either will lead to an overall reduction in mannosylated GPI species. Furthermore, to account for the observation that priming the cfs with GlcNAc-PI is significantly more efficient than priming with GlcN-PI, it has been postulated that substrate channeling occurs between the deNAc and MT1 (14,17). Given the structural similarity of the deNAc substrate GlcNAc-PI 1 and the MT1 substrate GlcN-PI 2, the analogs 11–19 may also interact with MT1. We assessed the specificity of the two most potent inhibitors 11 and 18 by measuring their ability to inhibit the cfs primed with GDP-[3H]Man and either the deNAc substrate GlcNAc-PI 1 or the MT1 substrate GlcN-PI 2. Compound 18 inhibited the formation of mannosylated products when the cfs was primed with GlcNAc-PI 1, but not when primed with GlcN-PI 2 (Figure 5A), suggesting that it inhibits only the deNAc. Compound 11 inhibited the formation of mannosylated products when the cfs was primed with either GlcNAc-PI 1 or GlcN-PI 2 (Figure 5A), suggesting either that it inhibits both the deNAc and MT1, or that it inhibits MT1 only. The apparent inhibition of MT1 by the glucocyclitol-phospholipid 11 may be due to substrate channeling between the deNAc and MT1, such that inhibition of the deNAc is able to prevent GlcN-PI from accessing the MT1 active site. However, the present data do not rule out direct inhibition of MT1 only.


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)

Inhibitors target the GlcNAc-PI de-N-acetylase. (A) Inhibition of the Trypanosoma brucei cell-free system by 11 and 18 (10 mm) when primed with either GlcNAc-PI or GlcN-PI. Conditions as Figure 4. (B) Inhibition of recombinant T. brucei GlcNAc-PI de-N-acetylase by 11 measured by electrospray tandem mass spectrometry. The intensity of the reaction product GlcN-IPC184 (m/z 672) is normalized to the turnover in uninhibited control. PI, phosphatidylinositol.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Inhibitors target the GlcNAc-PI de-N-acetylase. (A) Inhibition of the Trypanosoma brucei cell-free system by 11 and 18 (10 mm) when primed with either GlcNAc-PI or GlcN-PI. Conditions as Figure 4. (B) Inhibition of recombinant T. brucei GlcNAc-PI de-N-acetylase by 11 measured by electrospray tandem mass spectrometry. The intensity of the reaction product GlcN-IPC184 (m/z 672) is normalized to the turnover in uninhibited control. PI, phosphatidylinositol.
Mentions: The indirect cfs assay is unable to distinguish between the inhibition of the deNAc and inhibition of the first mannosyltransferase (MT1), because either will lead to an overall reduction in mannosylated GPI species. Furthermore, to account for the observation that priming the cfs with GlcNAc-PI is significantly more efficient than priming with GlcN-PI, it has been postulated that substrate channeling occurs between the deNAc and MT1 (14,17). Given the structural similarity of the deNAc substrate GlcNAc-PI 1 and the MT1 substrate GlcN-PI 2, the analogs 11–19 may also interact with MT1. We assessed the specificity of the two most potent inhibitors 11 and 18 by measuring their ability to inhibit the cfs primed with GDP-[3H]Man and either the deNAc substrate GlcNAc-PI 1 or the MT1 substrate GlcN-PI 2. Compound 18 inhibited the formation of mannosylated products when the cfs was primed with GlcNAc-PI 1, but not when primed with GlcN-PI 2 (Figure 5A), suggesting that it inhibits only the deNAc. Compound 11 inhibited the formation of mannosylated products when the cfs was primed with either GlcNAc-PI 1 or GlcN-PI 2 (Figure 5A), suggesting either that it inhibits both the deNAc and MT1, or that it inhibits MT1 only. The apparent inhibition of MT1 by the glucocyclitol-phospholipid 11 may be due to substrate channeling between the deNAc and MT1, such that inhibition of the deNAc is able to prevent GlcN-PI from accessing the MT1 active site. However, the present data do not rule out direct inhibition of MT1 only.

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