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Metabolomics and lipidomics reveal perturbation of sphingolipid metabolism by a novel anti-trypanosomal 3-(oxazolo[4,5-b]pyridine-2-yl)anilide

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Abstract

Introduction

Trypanosoma brucei is the causative agent of human African trypanosomiasis, which is responsible for thousands of deaths every year. Current therapies are limited and there is an urgent need to develop new drugs. The anti-trypanosomal compound, 3-(oxazolo[4,5-b]pyridine-2-yl)anilide (OXPA), was initially identified in a phenotypic screen and subsequently optimized by structure–activity directed medicinal chemistry. It has been shown to be non-toxic and to be active against a number of trypanosomatid parasites. However, nothing is known about its mechanism of action.

Objective

Here, we have utilized an untargeted metabolomics approach to investigate the biochemical effects and potential mode of action of this compound in T. brucei.

Methods

Total metabolite extracts were analysed by HILIC-chromatography coupled to high resolution mass spectrometry.

Results

Significant accumulation of ceramides was observed in OXPA-treated T. brucei. To further understand drug-induced changes in lipid metabolism, a lipidomics method was developed which enables the measurement of hundreds of lipids with high throughput and precision. The application of this LC–MS based approach to cultured bloodstream-form T. brucei putatively identified over 500 lipids in the parasite including glycerophospholipids, sphingolipids and fatty acyls, and confirmed the OXPA-induced accumulation of ceramides. Labelling with BODIPY-ceramide further confirmed the ceramide accumulation following drug treatment.

Conclusion

These findings clearly demonstrate perturbation of ceramide metabolism by OXPA and indicate that the sphingolipid pathway is a promising drug target in T. brucei.

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References

  • Ali, J. A., Creek, D. J., Burgess, K., Allison, H. C., Field, M. C., Maser, P., et al. (2013). Pyrimidine salvage in Trypanosoma brucei bloodstream forms and the trypanocidal action of halogenated pyrimidines. Molecular Pharmacology, 83(2), 439–453. doi:10.1124/mol.112.082321.

    Article  CAS  PubMed  Google Scholar 

  • Becker, G. W., & Lester, R. L. (1980). Biosynthesis of phosphoinositol-containing sphingolipids from phosphatidylinositol by a membrane preparation from Saccharomyces cerevisiae. Journal of Bacteriology, 142(3), 747–754.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Besteiro, S., Biran, M., Biteau, N., Coustou, V., Baltz, T., Canioni, P., et al. (2002). Succinate secreted by Trypanosoma brucei is produced by a novel and unique glycosomal enzyme, NADH-dependent fumarate reductase. Journal of Biological Chemistry, 277(41), 38001–38012. doi:10.1074/jbc.M201759200.

    Article  CAS  PubMed  Google Scholar 

  • Bromley, P. E., Li, Y. O., Murphy, S. M., Sumner, C. M., & Lynch, D. V. (2003). Complex sphingolipid synthesis in plants: characterization of inositolphosphorylceramide synthase activity in bean microsomes. Archives of Biochemistry and Biophysics, 417(2), 219–226.

    Article  CAS  PubMed  Google Scholar 

  • Brun, R., Blum, J., Chappuis, F., & Burri, C. (2010). Human African trypanosomiasis. The Lancet, 375(9709), 148–159.

    Article  Google Scholar 

  • Chai, X. (2014). Untargeted lipidomic profiling of human plasma reveals differences due to race, gender and smoking status. Metabolomics, 4(131), 2153. doi:10.4172/2153-0769.1000131.

    Google Scholar 

  • Chatterjee, A.K.N. A.S., Paraselli, P., Kondreddi, R.R., Leong, S.Y., Mishra, P.K., Moreau, R.J., Roland, J.T., Sim, W.L.S., Simon, O., Tan, L.J., Yeung, B.K., Zou, B., Bollu, V. (2014). Compounds and compositions for the treatment of parasitic diseases. US Patent 20140274926 A1. 18th September 2014.

  • Choi, J. Y., Podust, L. M., & Roush, W. R. (2014). Drug strategies targeting CYP51 in neglected tropical diseases. Chemical Reviews, 114(22), 11242–11271. doi:10.1021/cr5003134.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Creek, D. J., Anderson, J., McConville, M. J., & Barrett, M. P. (2012). Metabolomic analysis of trypanosomatid protozoa. Molecular and Biochemical Parasitology, 181(2), 73–84. doi:10.1016/j.molbiopara.2011.10.003.

    Article  CAS  PubMed  Google Scholar 

  • Creek, D. J., Jankevics, A., Breitling, R., Watson, D. G., Barrett, M. P., & Burgess, K. E. V. (2011). Toward global metabolomics analysis with hydrophilic interaction liquid chromatography-mass spectrometry: Improved metabolite identification by retention time prediction. Analytical Chemistry, 83(22), 8703–8710. doi:10.1021/ac2021823.

    Article  CAS  PubMed  Google Scholar 

  • Creek, D. J., Nijagal, B., Kim, D.-H., Rojas, F., Matthews, K. R., & Barrett, M. P. (2013). Metabolomics guides rational development of a simplified cell culture medium for drug screening against Trypanosoma brucei. Antimicrobial Agents and Chemotherapy, 57(6), 2768–2779. doi:10.1128/aac.00044-13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • de Koning, H. P. (2001). Transporters in African trypanosomes: Role in drug action and resistance. International Journal of Parasitology, 31(5–6), 512–522.

    Article  PubMed  Google Scholar 

  • Denny, P. W., Shams-Eldin, H., Price, H. P., Smith, D. F., & Schwarz, R. T. (2006). The protozoan inositol phosphorylceramide synthase: A novel drug target that defines a new class of sphingolipid synthase. Journal of Biological Chemistry, 281(38), 28200–28209. doi:10.1074/jbc.M600796200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • DNDi Oxaborole SCYX-7158 (HAT). http://www.dndi.org/diseases-projects/portfolio/oxaborole-scyx-7158.html. Accessed 05 June 2015.

  • Drexler, D. M., Reily, M. D., & Shipkova, P. A. (2011). Advances in mass spectrometry applied to pharmaceutical metabolomics. Analytical and Bioanalytical Chemistry, 399(8), 2645–2653. doi:10.1007/s00216-010-4370-8.

    Article  CAS  PubMed  Google Scholar 

  • Drugs for Neglected Diseases (2012). Pivotal Study of Fexinidazole for Human African Trypanosomiasis in Stage 2. http://www.clinicaltrials.gov/ct2/show/NCT01685827?term=fexinidazole&rank=3#wrapper. Accessed 23 Nov 2012.

  • Fairlamb, A., & Opperdoes, F. (1986). Carbohydrate metabolism in African trypanosomes, with special reference to the glycosome. In M. Morgan (Ed.), Carbohydrate Metabolism in Cultured Cells (pp. 183–224). US: Springer.

    Chapter  Google Scholar 

  • Ferrins, L., Rahmani, R., Sykes, M. L., Jones, A. J., Avery, V. M., Teston, E., et al. (2013). 3-(Oxazolo[4,5-b]pyridin-2-yl)anilides as a novel class of potent inhibitors for the kinetoplastid Trypanosoma brucei, the causative agent for human African trypanosomiasis. European Journal of Medicinal Chemistry, 66, 450–465. doi:10.1016/j.ejmech.2013.05.007.

    Article  CAS  PubMed  Google Scholar 

  • Fridberg, A., Olson, C. L., Nakayasu, E. S., Tyler, K. M., Almeida, I. C., & Engman, D. M. (2008). Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei. Journal of Cell Science, 121(Pt 4), 522–535. doi:10.1242/jcs.016741.

    Article  CAS  PubMed  Google Scholar 

  • Friedman, S., & Fraenkel, G. (1955). Reversible enzymatic acetylation of carnitine. Archives of Biochemistry and Biophysics, 59(2), 491–501.

    Article  CAS  PubMed  Google Scholar 

  • Futerman, A. H., & Hannun, Y. A. (2004). The complex life of simple sphingolipids. EMBO Reports, 5(8), 777–782. doi:10.1038/sj.embor.7400208.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gilbert, R. J., & Klein, R. A. (1984). Pyruvate kinase: A carnitine regulated site of ATP production in Trypanosoma brucei brucei. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 78(3), 595–599. doi:10.1016/0305-0491(84)90104-4.

    Article  CAS  Google Scholar 

  • Gilbert, R. J., Klein, R. A., & Johnson, P. (1983). Bromoacetyl-l-carnitine: Biochemical and antitrypanosomal actions against Trypanosoma brucei brucei. Biochemical Pharmacology, 32(22), 3447–3451. doi:10.1016/0006-2952(83)90375-1.

    Article  CAS  PubMed  Google Scholar 

  • Hanau, S., Rinaldi, E., Dallocchio, F., Gilbert, I. H., Dardonville, C., Adams, M. J., et al. (2004). 6-phosphogluconate dehydrogenase: a target for drugs in African trypanosomes. Current Medicinal Chemistry, 11(19), 2639–2650.

    Article  CAS  PubMed  Google Scholar 

  • Hu, C., van Dommelen, J., van der Heijden, R., Spijksma, G., Reijmers, T. H., Wang, M., et al. (2008). RPLC-ion-trap-FTMS method for lipid profiling of plasma: method validation and application to p53 mutant mouse model. Journal of Proteome Research, 7(11), 4982–4991. doi:10.1021/pr800373m.

    Article  CAS  PubMed  Google Scholar 

  • Kamleh, A., Barrett, M. P., Wildridge, D., Burchmore, R. J., Scheltema, R. A., & Watson, D. G. (2008). Metabolomic profiling using Orbitrap Fourier transform mass spectrometry with hydrophilic interaction chromatography: A method with wide applicability to analysis of biomolecules. Rapid Communications in Mass Spectrometry, 22(12), 1912–1918. doi:10.1002/rcm.3564.

    Article  CAS  PubMed  Google Scholar 

  • Matthews, K. R. (2005). The developmental cell biology of Trypanosoma brucei. Journal of Cell Science, 118(2), 283–290. doi:10.1242/jcs.01649.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mina, J. G., Pan, S.-Y., Wansadhipathi, N. K., Bruce, C. R., Shams-Eldin, H., Schwarz, R. T., et al. (2009). The Trypanosoma brucei sphingolipid synthase, an essential enzyme and drug target. Molecular and Biochemical Parasitology, 168(1), 16–23. doi:10.1016/j.molbiopara.2009.06.002.

    Article  CAS  PubMed  Google Scholar 

  • Nok, A. J. (2003). Arsenicals (melarsoprol), pentamidine and suramin in the treatment of human African trypanosomiasis. Parasitology Research, 90(1), 71–79. doi:10.1007/s00436-002-0799-9.

    PubMed  Google Scholar 

  • Patnaik, P. K., Field, M. C., Menon, A. K., Cross, G. A. M., Yee, M. C., & Bütikofer, P. (1993). Molecular species analysis of phospholipids from Trypanosoma brucei bloodstream and procyclic forms. Molecular and Biochemical Parasitology, 58(1), 97–105. doi:10.1016/0166-6851(93)90094-E.

    Article  CAS  PubMed  Google Scholar 

  • Priotto, G., Kasparian, S., Mutombo, W., Ngouama, D., Ghorashian, S., Arnold, U., et al. (2009). Nifurtimox-eflornithine combination therapy for second-stage African Trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase III, non-inferiority trial. The Lancet, 374(9683), 56–64.

    Article  CAS  Google Scholar 

  • Richmond, G. S., Gibellini, F., Young, S. A., Major, L., Denton, H., Lilley, A., et al. (2010). Lipidomic analysis of bloodstream and procyclic form Trypanosoma brucei. Parasitology, 137(9), 1357–1392. doi:10.1017/s0031182010000715.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schenkman, S., Jiang, M. S., Hart, G. W., & Nussenzweig, V. (1991). A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells. Cell, 65(7), 1117–1125.

    Article  CAS  PubMed  Google Scholar 

  • Seebeck, T., & Maser, P. (2009). Drug resistance in African Trypanosomiasis. In D. L. Mayers (Ed.), Antimicrobial drug resistance (pp. 589–604). Newyork: Humana Press.

    Chapter  Google Scholar 

  • Serricchio, M., & Butikofer, P. (2011). Trypanosoma brucei: a model micro-organism to study eukaryotic phospholipid biosynthesis. FEBS Journal, 278(7), 1035–1046. doi:10.1111/j.1742-4658.2011.08012.x.

    Article  CAS  PubMed  Google Scholar 

  • Silva, A. M., Cordeiro-da-Silva, A., & Coombs, G. H. (2011). Metabolic variation during development in culture of Leishmania donovani promastigotes. PLoS Neglected Tropical Diseases, 5(12), e1451. doi:10.1371/journal.pntd.0001451.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Simarro, P. P., Diarra, A., Ruiz Postigo, J. A., Franco, J. R., & Jannin, J. G. (2011). The human African trypanosomiasis control and surveillance programme of the World Health Organization 2000–2009: The way forward. PLoS Neglected Tropical Diseases, 5(2), e1007. doi:10.1371/journal.pntd.0001007.

    Article  PubMed  PubMed Central  Google Scholar 

  • Sutterwala, S. S., Creswell, C. H., Sanyal, S., Menon, A. K., & Bangs, J. D. (2007). De novo sphingolipid synthesis is essential for viability, but not for transport of glycosylphosphatidylinositol-anchored proteins, in African trypanosomes. Eukaryotic Cell, 6(3), 454–464. doi:10.1128/EC.00283-06.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sutterwala, S. S., Hsu, F. F., Sevova, E. S., Schwartz, K. J., Zhang, K., Key, P., et al. (2008). Developmentally regulated sphingolipid synthesis in African trypanosomes. Molecular Microbiology, 70(2), 281–296. doi:10.1111/j.1365-2958.2008.06393.x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sykes, M. L., & Avery, V. M. (2009). Development of an Alamar Blue viability assay in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427. American Journal of Tropical Medicine and Hygeine, 81(4), 665–674. doi:10.4269/ajtmh.2009.09-0015.

    Article  CAS  Google Scholar 

  • Sykes, M. L., Baell, J. B., Kaiser, M., Chatelain, E., Moawad, S. R., Ganame, D., et al. (2012). Identification of compounds with anti-proliferative activity against Trypanosoma brucei brucei strain 427 by a whole cell viability based HTS campaign. PLoS Neglected Tropical Diseases, 6(11), e1896. doi:10.1371/journal.pntd.0001896.

    Article  PubMed  PubMed Central  Google Scholar 

  • Tielens, A. G. M., & Van Hellemond, J. J. (1998). Differences in energy metabolism between Trypanosomatidae. Parasitology Today, 14(7), 265–272. doi:10.1016/S0169-4758(98)01263-0.

    Article  CAS  PubMed  Google Scholar 

  • t’Kindt, R., Scheltema, R. A., Jankevics, A., Brunker, K., Rijal, S., Dujardin, J.-C., Breitling, R., et al. (2010). Metabolomics to unveil and understand phenotypic diversity between pathogen populations. PLoS Neglected Tropical Diseases. 4(11), e904. doi:10.1371/journal.pntd.0000904.

    Article  PubMed  PubMed Central  Google Scholar 

  • Trochine, A., Creek, D. J., Faral-Tello, P., Barrett, M. P., & Robello, C. (2014). Benznidazole biotransformation and multiple targets in Trypanosoma cruzi revealed by metabolomics. PLoS Neglected Tropical Diseases, 8(5), e2844. doi:10.1371/journal.pntd.0002844.

    Article  PubMed  PubMed Central  Google Scholar 

  • Vincent, I. M., Creek, D. J., Burgess, K., Woods, D. J., Burchmore, R. J., & Barrett, M. P. (2012). Untargeted metabolomics reveals a lack of synergy between nifurtimox and eflornithine against Trypanosoma brucei. PLoS Neglected Tropical Diseases, 6(5), e1618. doi:10.1371/journal.pntd.0001618.

    Article  PubMed  PubMed Central  Google Scholar 

  • Voogd, T. E., Vansterkenburg, E. L., Wilting, J., & Janssen, L. H. (1993). Recent research on the biological activity of suramin. Pharmacological Reviews, 45(2), 177–203.

    CAS  PubMed  Google Scholar 

  • Yamada, T., Uchikata, T., Sakamoto, S., Yokoi, Y., Fukusaki, E., & Bamba, T. (2013). Development of a lipid profiling system using reverse-phase liquid chromatography coupled to high-resolution mass spectrometry with rapid polarity switching and an automated lipid identification software. Journal of Chromatography A, 1292, 211–218.

    Article  CAS  PubMed  Google Scholar 

  • Young, S., & Smith, T. K. (2010). The essential neutral sphingomyelinase is involved in the trafficking of the variant surface glycoprotein in the bloodstream form of Trypanosoma brucei. Molecular Microbiology, 76(6), 1461–1482. doi:10.1111/j.1365-2958.2010.07151.x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

DJC acknowledges support from a NHMRC training fellowship. LF acknowledges an Australian Postgraduate Award. MJM is an NHMRC Principal Research Fellow. Financial support was received from NHMRC project Grants APP1025581 and APP1067728.

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Authors and Affiliations

  1. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, 381 Royal Parade, Parkville, Victoria, 3052, Australia

    Daniel Stoessel, Katherine M. Ellis & Darren J. Creek

  2. Department of Biotechnology, Beuth University of Applied Sciences, Berlin, Germany

    Daniel Stoessel

  3. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia

    Cameron J. Nowell

  4. Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, Nathan, QLD, Australia

    Amy J. Jones & Vicky M. Avery

  5. Department of Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, 381 Royal Parade, Parkville, Victoria, 3052, Australia

    Lori Ferrins, Raphael Rahmani & Jonathan B. Baell

  6. Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK

    Jennifer Riley & Kevin D. Read

  7. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Australia

    Malcolm J. McConville

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  1. Daniel Stoessel
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Corresponding authors

Correspondence to Jonathan B. Baell or Darren J. Creek.

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Electronic supplementary material

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S1

IDEOM metabolite list and metadata from HILIC metabolomics study (XLSB 31541 kb)

S2

IDEOM metabolite list and metadata from lipidomics study (XLSB 23466 kb)

S3

MSMS spectra of significant ceramides and acetylcarnitine (PDF 375 kb)

S4

Results from IC50 analysis in presence of Carnitine (PDF 38 kb)

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Stoessel, D., Nowell, C.J., Jones, A.J. et al. Metabolomics and lipidomics reveal perturbation of sphingolipid metabolism by a novel anti-trypanosomal 3-(oxazolo[4,5-b]pyridine-2-yl)anilide. Metabolomics 12, 126 (2016). https://doi.org/10.1007/s11306-016-1062-1

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