Chemical validation of GPI biosynthesis as a drug target against African sleeping sickness

Division of Biological Chemistry and Molecular Microbiology, The Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee, Scotland, UK.
The EMBO Journal (Impact Factor: 10.75). 12/2004; 23(23):4701-8. DOI: 10.1038/sj.emboj.7600456
Source: PubMed

ABSTRACT It has been suggested that compounds affecting glycosylphosphatidylinositol (GPI) biosynthesis in bloodstream form Trypanosoma brucei should be trypanocidal. We describe cell-permeable analogues of a GPI intermediate that are toxic to this parasite but not to human cells. These analogues are metabolized by the T. brucei GPI pathway, but not by the human pathway. Closely related nonmetabolizable analogues have no trypanocidal activity. This represents the first direct chemical validation of the GPI biosynthetic pathway as a drug target against African human sleeping sickness. The results should stimulate further inhibitor design and synthesis and encourage the search for inhibitors in natural product and synthetic compound libraries.

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Available from: Michael A J Ferguson, Sep 21, 2014
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    • "GPI anchors are ubiquitous to eukaryotes and comprise of the basic core structure of NH 2 CH 2 CH 2 PO 4 H-6Manα1- 2Manα1-6Manα1-4GlcNα1-6D-myo-inositol-1-HPO 4 -lipid (EtN-P-Man 3 GlcN-PI), with a lipid moiety of diacylglycerol, alkylacylglycerol, or ceramide [8]. Previously, the biosynthesis of GPI anchors in bloodstream form T. brucei has been both genetically and chemically validated as a therapeutic drug target [9] [10] [11]. "
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    ABSTRACT: Inositol-3-phosphate synthase (INO1) has previously been genetically validated as a drug target against Trypanosoma brucei, the causative agent of African sleeping sickness. Chemical intervention of this essential enzyme could lead to new therapeutic agents. Unfortunately, no potent inhibitors of INO1 from any organism have been reported, so a screen for potential novel inhibitors of T. brucei INO1was undertaken. Detection of inhibition of T. brucei INO1 is problematic due to the nature of the reaction. Direct detection requires differentiation between glucose-6-phosphate and inositol-3-phosphate. Coupled enzyme assays could give false positives as potentially they could inhibit the coupling enzyme. Thus, an alternative approach of differential scanning fluorimetry to identify compounds that interact with T. brucei INO1 was employed to screen ~670 compounds from the MayBridge Rule of 3 Fragment Library. This approach identified 38 compounds, which significantly altered the Tm of TbINO1. Four compounds showed trypanocidal activity with ED50s in the tens of micromolar range, with 2 having a selectivity index in excess of 250. The trypanocidal and general cytotoxicity activities of all of the compounds in the library are also reported, with the best having ED50S of ~20 μM against T. brucei.
    05/2011; 2011:389364. DOI:10.4061/2011/389364
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    • "GPIno provides the anchoring hydrophobic moiety of the GPI (Masterson et al., 1989) and GPEth donates phosphoethanolamine (Menon et al., 1993). As GPIno and GPEth levels are also decreased after ACP knockout in BSF trypanosomes (T.K. Smith, unpubl.), a drug targeting mitochondrial fatty acid synthesis could indirectly diminish the BSFs ability to evade the host immune system through a detrimental effect on GPI anchor biosynthesis required for cell surface expression of the variant surface glycoprotein (Nagamune et al., 2000; Chang et al., 2002; Smith et al., 2004). There are several classical inhibitors that target fatty acid synthesis, some of which have been tested on trypanosomes with varying efficacies (Morita et al., 2000; Paul et al., 2004; Jones et al., 2005). "
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    ABSTRACT: Trypanosoma brucei use microsomal elongases for de novo synthesis of most of its fatty acids. In addition, this parasite utilizes an essential mitochondrial type II synthase for production of octanoate (a lipoic acid precursor) as well as longer fatty acids such as palmitate. Evidence from other organisms suggests that mitochondrially synthesized fatty acids are required for efficient respiration but the exact relationship remains unclear. In procyclic form trypanosomes, we also found that RNAi depletion of the mitochondrial acyl carrier protein, an important component of the fatty acid synthesis machinery, significantly reduces cytochrome-mediated respiration. This reduction was explained by RNAi-mediated inhibition of respiratory complexes II, III and IV, but not complex I. Other effects of RNAi, such as changes in mitochondrial morphology and alterations in membrane potential, raised the possibility of a change in mitochondrial membrane composition. Using mass spectrometry, we observed a decrease in total and mitochondrial phosphatidylinositol and mitochondrial phosphatidylethanolamine. Thus, we conclude that the mitochondrial synthase produces fatty acids needed for maintaining local phospholipid levels that are required for activity of respiratory complexes and preservation of mitochondrial morphology and function.
    Molecular Microbiology 04/2008; 67(5):1125-42. DOI:10.1111/j.1365-2958.2008.06112.x · 5.03 Impact Factor
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    • "It has previously been shown that GPI anchor biosynthesis is essential for the survival of bloodstream-form T. brucei (Nagamune et al., 2000; Chang et al., 2002; Smith et al., 2004); we surmized that in light of this, INO1 and hence the de novo synthesis of myo-inositol would also be essential if they were responsible for providing myo-inositol for synthesis of PI for GPI anchors. We were unable to replace both allelic copies of INO1 to create a null mutant, suggesting that TbINO1 may be an essential gene and surprisingly, the addition of extra myo-inositol (from 40 mM to 100 mM) to the media did not enable the creation of a null mutant. "
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    ABSTRACT: In bloodstream-form Trypanosoma brucei (the causative agent of African sleeping sickness) the glycosylphosphatidylinositol (GPI) anchor biosynthetic pathway has been validated genetically and chemically as a drug target. The conundrum that GPI anchors could not be in vivo labelled with [3H]-inositol led us to hypothesize that de novo synthesis was responsible for supplying myo-inositol for phosphatidylinositol (PI) destined for GPI synthesis. The rate-limiting step of the de novo synthesis is the isomerization of glucose 6-phosphate to 1-D-myo-inositol-3-phosphate, catalysed by a 1-D-myo-inositol-3-phosphate synthase (INO1). When grown under non-permissive conditions, a conditional double knockout demonstrated that INO1 is an essential gene in bloodstream-form T. brucei. It also showed that the de novo synthesized myo-inositol is utilized to form PI, which is preferentially used in GPI biosynthesis. We also show for the first time that extracellular myo-inositol can in fact be used in GPI formation although to a limited extent. Despite this, extracellular inositol cannot compensate for the deletion of INO1. Supporting these results, there was no change in PI levels in the conditional double knockout cells grown under non-permissive conditions, showing that perturbation of growth is due to a specific lack of de novo synthesized myo-inositol and not a general inositol-less death. These results suggest that there is a distinction between de novo synthesized myo-inositol and that from the extracellular environment.
    Molecular Microbiology 08/2006; 61(1):89-105. DOI:10.1111/j.1365-2958.2006.05216.x · 5.03 Impact Factor
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