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: 10.43). 12/2004; 23(23):4701-8. DOI: 10.1038/sj.emboj.7600456
Source: PubMed


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.
    Full-text · Article · May 2011
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    • "In T. brucei, the GPI biosynthesis has already been validated as a molecular target for development of new drugs against African sleeping sickness (Smith et al, 2004). To exploit GPIs as targets for the development of new therapies against other endemic protozoa (e.g., T. cruzi, P. falciparum, Leishmania spp.), a detailed, large-scale analysis of the GPI-anchored molecules expressed on the cell surface of these parasites (i.e., the GPIome) is of paramount importance. "
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    ABSTRACT: Glycosylphosphatidylinositol (GPI) anchoring is a common, relevant posttranslational modification of eukaryotic surface proteins. Here, we developed a fast, simple, and highly sensitive (high attomole-low femtomole range) method that uses liquid chromatography-tandem mass spectrometry (LC-MS(n)) for the first large-scale analysis of GPI-anchored molecules (i.e., the GPIome) of a eukaryote, Trypanosoma cruzi, the etiologic agent of Chagas disease. Our genome-wise prediction analysis revealed that approximately 12% of T. cruzi genes possibly encode GPI-anchored proteins. By analyzing the GPIome of T. cruzi insect-dwelling epimastigote stage using LC-MS(n), we identified 90 GPI species, of which 79 were novel. Moreover, we determined that mucins coded by the T. cruzi small mucin-like gene (TcSMUG S) family are the major GPI-anchored proteins expressed on the epimastigote cell surface. TcSMUG S mucin mature sequences are short (56-85 amino acids) and highly O-glycosylated, and contain few proteolytic sites, therefore, less likely susceptible to proteases of the midgut of the insect vector. We propose that our approach could be used for the high throughput GPIomic analysis of other lower and higher eukaryotes.
    Full-text · Article · Feb 2009 · Molecular Systems Biology
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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.
    Preview · Article · Apr 2008 · Molecular Microbiology
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