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Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach

Nature Chemistry volume 6, pages 112–121 (2014)Cite this article

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Abstract

Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approximately one million deaths per annum worldwide. Chemical validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyses the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, we report an integrated chemical biology approach to explore protein myristoylation in the major human parasite P. falciparum, combining chemical proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-molecule N-myristoyltransferase inhibitors. We demonstrate that N-myristoyltransferase is an essential and chemically tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a critical subcellular organelle in the parasite life cycle. Our studies provide the basis for the development of new antimalarials targeting N-myristoyltransferase.

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Figure 1: YnMyr-CoA is an effective substrate mimic for P. falciparum and P. vivax N-myristoyltransferases.
Figure 2: YnMyr tags N-myristoylated and GPI-anchored proteins in blood-stage P. falciparum.
Figure 3: Chemical proteomic discovery and direct detection of modification site for N-myristoylated and GPI-anchored proteins in blood-stage P. falciparum.
Figure 4: Compounds 1a–c and 2a–b bind in the Plasmodium NMT peptide binding pocket and inhibit the recombinant enzyme.
Figure 5: Inhibition of NMT in P. falciparum blood-stage parasites leads to antiparasitic activity, and reduces parasitaemia in an in vivo model of malaria.
Figure 6: NMT inhibition results in loss of the IMC and irreversible loss of parasite viability before parasite egress.

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Acknowledgements

The authors thank the staff at the Diamond Light Source (Harwell, UK) for assistance with crystallography, and S. Roberts for crystal handling. The authors also thank L. Haigh for assistance with mass spectrometry and P. Bowyer and members of the Tate group for critical reading of the manuscript. The authors thank P. Wyatt for helpful discussions. This work was supported by grants from the Institute of Chemical Biology (Imperial College London), the UK Engineering and Physical Sciences Research Council (Studentship awards and Doctoral Prize Fellowship awards to M.H.W. and M.D.R., grants EP/F500416/1 and EP/K039946/1), the UK Medical Research Council (grants G0900278, MR/K011782/1 and U117532067), European Commission FP7 (2007–2013) (grant 242095), the German Research Foundation (DFG, grant BR 4387/1-1) and the UK Biotechnology and Biological Sciences Research Council (grant BB/D02014X/1).

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  1. William P. Heal & Robin J. Leatherbarrow

    Present address: Present addresses: Department of Chemistry, Kings College London, London SE1 1UL, UK (W.P.H.); Liverpool John Moores University, Liverpool L1 2UA, UK (R.J.L.),

Authors and Affiliations

  1. Department of Chemistry, Imperial College London, London, SW7 2AZ, UK

    Megan H. Wright, Mark D. Rackham, William P. Heal, Malgorzata Broncel, Remigiusz A. Serwa, Robin J. Leatherbarrow & Edward W. Tate

  2. Institute of Chemical Biology, Imperial College London, London, SW7 2AZ, UK

    Megan H. Wright, David J. Mann, Robin J. Leatherbarrow & Edward W. Tate

  3. Division of Parasitology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK

    Barbara Clough, Kaveri Rangachari, Munira Grainger, David K. Moss & Anthony A. Holder

  4. Department of Chemistry, York Structural Biology Laboratory, University of York, York, YO10 5DD, UK

    James A. Brannigan & Anthony J. Wilkinson

  5. Protein and Nucleic Acid Chemistry Laboratory, University of Leicester, Hodgkin Building, Lancaster Road, Leicester, LE1 9HN, UK

    Andrew R. Bottrill

  6. Centre for Genetics and Genomics, School of Life Sciences, Queens Medical Centre, University of Nottingham, Nottingham, NG2 7UH, UK

    Declan Brady & Rita Tewari

  7. Division of Molecular Biosciences, Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK

    David J. Mann

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Contributions

M.H.W. designed and executed the chemical proteomics experiments, performed experiments on parasite samples and analysed data. M.G., D.K.M., K.R. and B.C. produced synchronized parasites and performed parasite culture. D.K.M., B.C. and K.R. produced protein-specific antibodies and K.R. and B.C. performed immunofluorescence microscopy under the guidance of A.A.H. M.D.R., E.W.T. and R.J.L. designed series 2 and M.D.R. synthesized compounds 2a–b. J.A.B. prepared recombinant proteins, performed crystallization experiments and determined X-ray structures, in collaboration with A.J.W. A.R.B. assisted M.H.W. with aspects of proteomic data generation and analysis. W.P.H. and R.A.S. synthesized compounds 1a–c. M.B., E.W.T. and R.A.S. designed and synthesized reagent AzKTB. R.A.S., E.W.T. and M.H.W. performed nanoLC-MS/MS experiments, proteomic identification of modified peptides and whole proteome analyses. R.T. and D.B. performed in vivo analysis using the rodent malaria model. E.W.T. conceived and designed experiments, analysed data and directed the overall collaboration. M.H.W., A.A.H. and E.W.T. co-wrote the manuscript, with comments and contributions from all authors.

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Correspondence to Edward W. Tate.

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Supplementary information

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Supplementary information (PDF 2884 kb)

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Table 1: Prediction of N-myristoylation by bioinformatic tools (XLSX 37 kb)

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Table 3: Total proteomic datasets (XLSX 25 kb)

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Table 9: Changes in protein abundance due to NMT inhibition (XLSX 174 kb)

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Wright, M., Clough, B., Rackham, M. et al. Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach. Nature Chem 6, 112–121 (2014). https://doi.org/10.1038/nchem.1830

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