Molecular parasitology, glycobiology and drug discovery

Research

Insect-transmitted protozoan parasites cause widespread and debilitating diseases in man and domestic livestock throughout the tropics. Examples of diseases caused by trypanosomatid parasites include African sleeping sickness (caused by Trypanosoma brucei and transmitted by tsetse flies), Chagas disease (caused by Trypanosoma cruzi) and kala-azar, espundia and oriental sore (caused by the Leishmania). There are no vaccines against these diseases and most of the available drug treatments are toxic and/or ineffective.

Parasite surface molecules must protect the organisms and enable them to identify, and interact with, cells of both the insect vector and the animal host. Many trypanosomatid parasite surface molecules are either glycosylphosphatidylinositol (GPI) anchored glycoproteins or GPI-related glycolipids (Fig.1).

 The parasite GPI biosynthetic pathway, and the pathways that assemble the sugar nucleotides that fuel it and the protein O- and N-glycosylation pathways, are validated targets for the development of new chemotherapeutic agents.

Our research is multi-disciplinary and involves defining:

• The "structural repertoire" of the parasite glycoproteins (Figs.1 & 3)
• The "biosynthetic repertoire" of necessary glycosyltransferases and  processing enzymes needed to create the structural repertoire (Fig 2)
• The "metabolic repertoire" of sugar nucleotides, and their biosynthetic and transporter proteins, needed to fuel the biosynthetic repertoire (Figs.2 & 4)

These goals involve:

(A) The isolation and analysis of parasite surface molecules and sugar nucleotide metabolites using advanced mass spectrometric methods (1-3).

(B) Bioinformatics (in collaboration with Geoff Barton), proteomics, gene-knockout and cell biology and advanced mass spectrometric methods, to identify, localise and study the functions of glycoprotein (GPI anchoring and protein N-glycosylation) and sugar nucleotide biosynthetic enzymes (4-9).

(C) Synthetic organic chemistry (including collaboration with Andrei Nikolaev) and enzymology to define the properties and substrate specificities of enzymes involved in protein glycosylation, GPI anchor biosynthesis and sugar nucleotide assembly (10-13).

(D) Drug Discovery, involving X-ray crystallography and molecular modelling of drug target enzymes (8, 9, 14, 15) (Fig.5) (in collaboration with Bill Hunter, Daan van Aalten and the Structural Genomics Consortium), computational chemistry (in collaboration with Ruth Brenk), high-throughput screening and molecular pharmacology (in collaboration with David Gray) and medicinal chemistry (in collaboration with Ian Gilbert and Paul Wyatt).

We also have ongoing studies on the phosphoproteome of T.brucei (16-18)

Our ultimate aim is to discover new anti-parasite therapeutic agents for clinical trials through our unique http://www.drugdiscovery.dundee.ac.uk/Drug Discovery Unit (17).

Biomarker Discovery

In addition to our work on parasite glycobiology, we use our expertise in mass spectrometry and proteomics (18) to collaborate with clinical and biomedical colleagues, like Professor Helen Colhoun, Professor Bob Steele, Dr John Dillon and Professor Roland Wolf to discover and validate biomarkers of complications of diabetes, colon cancer and early markers of drug toxicity and non-alcoholic fatty liver disease.

Publications

1. Atrih, A., Richardson, J.M., Prescott, A.R. and Ferguson, M.A.J. (2005) Trypanosoma brucei glycoproteins contain novel giant poly-N-acetyl-lactosamine  carbohydrate chains. J. Biol. Chem. 280, 865-871.
2. Mehlert, A., Sullivan, L. and Ferguson, M.A.J. (2010) Glycotyping of Trypanosoma brucei variant surface glycoprotein MITat1.8. Mol. Biochem. Parasitol. 174, 74-77.
3. Turnock, D.C. and Ferguson, M.A.J. (2007) The sugar nucleotide pools of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. Eukaryotic Cell 6, 1450-1463.
4. Stokes, M.J., Güther, M.L.S., Turnock, D.C., Martin, K.L., Alphey, M.S. and Ferguson, M.A.J. (2008) The synthesis of UDP-N-acetylglucosamine is essential for bloodstream form Trypanosoma brucei in vitro and in vivo and UDP-N-acetylglucosamine starvation reveals a hierarchy in parasite protein glycosylation. J.Biol.Chem. 283, 16147-16161.
5. Izquierdo, L., Nakanishi, M, Mehlert, A., Machray, G., Barton, G.J. and Ferguson, M.A.J. (2009) Identification of a GPI-anchor modifying β1-3 N-acetylglucosaminyltransferase in Trypanosoma brucei. Mol. Microbiol. 71, 478-491
6. Izquierdo, L., Schulza, B.L., Rodrigues, J.A., Güther, M.L.S., Proctor, J.B., Barton, G.J., Aebi, M. and Ferguson, M.A.J. (2009) Distinct oligosaccharide donor and peptide acceptor specificities of Trypansosoma brucei oligosaccharyltransferases. EMBO J. 28, 2650-2661.
7. Güther, M.L.S., Beattie, K., Lamont, D.J., James, J., Prescott, A.R. and Ferguson, M.A.J. (2009) The fate of GPI-less procyclin and characterisation of sialylated non-GPI anchored surface coat molecules of procyclic form Trypanosoma brucei. Eukaryotic Cell, 8, 1407-1417.
8. Marino, K., Güther, M.L., Wernimont, A., Amani, M., Hui, R. and Ferguson, M.A.J. (2010)
   Identification, subcellular localization, biochemical properties and high-resolution crystal structure of Trypanosoma brucei UDP-glucose pyrophosphory. Glycobiology, 12, 1619-1630.
9.  Mariño, K.M.L., Güther, M.L., Wernimont, A.K., Hui, R., Ferguson, M.A. (2011) “Characterization, localization, essentiality and high-resolution crystal structure of Glucosamine 6-phosphate N-Acetyltranserase from Trypanosoma brucei”. Eukaryot. Cell 10 (7), 985-997
10. Crossman, A.T., Urbaniak, M.D. and Ferguson, M.A.J. (2008) Synthesis of 1-D-6-0- (2-N-hydroxyurea-2-deoxy-α-D-glucopyranosyl)-myo-inositol-(octadecyl phosphate): a potential metalloenzyme inhibitor of glycosylphatidylinositol biosynthesis. Carbohyd.Res. 343, 1478-1481.
11. Urbaniak, M.D., Crossman, A. and Ferguson, M.A.J. (2008) Probing Trypanosoma brucei glycosylphosphatidylinositol biosynthesis using novel precursor-analogues. Chemical Biology & Drug Design 72, 127-132.
12. Urbaniak, M.D., Yashunsky, D.V., Crossman, A., Nikolaev, A.V. and Ferguson, M.A.J. (2008) Probing enzymes late in the trypanosomal glycosylphosphatidylinositol biosynthetic pathway with synthetic glycosylphosphatidylinositol analogues. ACS Chemical Biology 3, 625-634.
13. Sizova, O. V., A. J. Ross, Ivanova, I. A., Borodkin, V. S., Ferguson, M. A. J., Nikolaev, A. V. (2011). "Probing Elongating and Branching beta-d-Galactosyltransferase Activities in Leishmania Parasites by Making Use of Synthetic Phosphoglycans." ACS Chem Biol 6 (6), 648-657
14. Alphey, M.S., Barton, A., Urbaniak, M.D., Boons, G-J., Ferguson, M.A.J. and Hunter, W.N. (2006) Trypanosoma brucei UDP-galactose-4'-epimerase in ternary complex with NAD+ and the substrate analogue UDP-4-deoxy-4-fluoro-α-D-galactose. Acta Cryst, F62, 829-834.
15. Urbaniak, M.D., Tabudravu, J.N., Msaki, A., Mansfield Matera, K., Brenk, R., Jaspers, M. and Ferguson, M.A.J. (2006) Identification of Novel Inhibitors of UDP-Glc 4'-Epimerase, a Validated Drug Target for African Sleeping Sickness. Bioorg. Med.Chem. Letters 16, 5744-5747.
16. Nett, I.R.E., Davidson, L., Lamont, D.J. and Ferguson, M.A.J. (2009) Trypanosoma brucei: Identification and specific localization of tryosine phosphorylated proteins. Eukaryotic Cell 8: 617-626.
17. Nett, I.R.E., Martin, D.M.A., Miranda-Saavedra, D., Lamont, D.J., Barber, J.D., Mehlert, A. and Ferguson, M.A.J. (2009) The phosphoproteome of bloodstream form Trypanonosoma brucei, causative agent of African Sleeping Sickness. Mol. Cell. Proteomics 8, 1527-1538.
18. Martin, D.M.A., Nett, I.R.E., Barber, J.D., and Ferguson, M.A.J. (2010) Prophossi: Automating Expert Validation of Phosphopeptide-Spectrum Matches from Tandem Mass Spectrometry. Bioinformatics, 26, 2153-2159
19. Frearson, J.F., Brand,S., McElroy, S.P., Cleghorn, L.A.T., Smid, O., Stojanovski, L., Price, H.P., Guther, M.L.S., Torrie, L.S., Robinson, D.A., Hallyburton, I., Mpamhanga, C.P. Brannigan, J.A. Wilkinson, A.J., Hodgkinson, M. Hui, R., Qui, W. Raimi, O.G. van Aalten, D.M.F., Brenk, R., Gilbert, I.H., Read, K.D., Fairlamb, A.H.Ferguson, M.A.J., Smith, D.F. and Wyatt, P.G (2010) N-Myristoyltransferase inhibitors: new leads for the treatment of human African trypanosomiasis. Nature 464, 728-732.
20. Atrih, A., Turnock, D., Sellar, G., Thompson, A., Feuerstein, G., Ferguson, M.A.J and Huang, J T-J (2010) Stoichiometric Quantification of Akt Phosphorylation using LC-MS/MS. J. Proteome Res. 9, 743-751.