Abstract

Malaria control is heavily dependent on chemotherapeutic agents for disease prevention and drug treatment. Defining the mechanism of action for licensed drugs, for which no target is characterized, is critical to the development of their second-generation derivatives to improve drug potency towards inhibition of their molecular targets. Mefloquine is a widely used antimalarial without a known mode of action. Here, we demonstrate that mefloquine is a protein synthesis inhibitor. We solved a 3.2 Å cryo-electron microscopy structure of the Plasmodium falciparum 80S ribosome with the (+)-mefloquine enantiomer bound to the ribosome GTPase-associated centre. Mutagenesis of mefloquine-binding residues generates parasites with increased resistance, confirming the parasite-killing mechanism. Furthermore, structure-guided derivatives with an altered piperidine group, predicted to improve binding, show enhanced parasiticidal effect. These data reveal one possible mode of action for mefloquine and demonstrate the vast potential of cryo-electron microscopy to guide the development of mefloquine derivatives to inhibit parasite protein synthesis.

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References

  1. 1.

    World Malaria Report (WHO, 2016);

  2. 2.

    et al. Malaria. Lancet 383, 723–735 (2014).

  3. 3.

    , & Malaria medicines: a glass half full? Nat. Rev. Drug Discov. 14, 424–442 (2015).

  4. 4.

    & Neuropsychiatric adverse reactions to mefloquine: a systematic comparison of prescribing and patient safety guidance in the US, UK, Ireland, Australia, New Zealand, and Canada. Neurol. Ther. 5, 69–83 (2016).

  5. 5.

    et al. Decreasing pfmdr1 copy number suggests that Plasmodium falciparum in western Cambodia is regaining in vitro susceptibility to mefloquine. Antimicrob. Agents Chemother. 59, 2934–2937 (2015).

  6. 6.

    Malaria wars. Sci. Transl. Med. 352, 398–405 (2016).

  7. 7.

    , , & Polymorphisms within PfMDR1 alter the substrate specificity for anti-malarial drugs in Plasmodium falciparum. Mol. Microbiol. 70, 786–798 (2008).

  8. 8.

    , & Selection for mefloquine resistance in Plasmodium falciparum is linked to amplification of the pfmdr1 gene and cross-resistance to halofantrine and quinine. Proc. Natl Acad. Sci. USA 91, 1143–1147 (1994).

  9. 9.

    , , , . & Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 403, 906–909 (2000).

  10. 10.

    , , & Transporters as mediators of drug resistance in Plasmodium falciparum. Int. J. Parasitol. 40, 1109–1118 (2010).

  11. 11.

    et al. Thousands of chemical starting points for antimalarial lead identification. Nature 465, 305–310 (2010).

  12. 12.

    et al. Tetracyclines specifically target the apicoplast of the malaria parasite Plasmodium falciparum. Antimicrob. Agents Chemother. 50, 3124–3131 (2006).

  13. 13.

    , & The effects of anti-bacterials on the malaria parasite Plasmodium falciparum. Mol. Biochem. Parasitol. 152, 181–191 (2007).

  14. 14.

    et al. Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine. eLife 3, e03080 (2014).

  15. 15.

    et al. The structure of the eukaryotic ribosome at 3.0 Å resolution. Science 334, 1524–1529 (2011).

  16. 16.

    et al. Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation. EMBO J. 23, 1008–1019 (2004).

  17. 17.

    et al. The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell 103, 1143–1154 (2000).

  18. 18.

    , & Antimalarials. 7. Bis(trifluoromethyl)-α-(2-piperidyl)-4-quinolinemethanols. J. Med. Chem. 14, 926–928 (1971).

  19. 19.

    et al. Translational regulation via L11: molecular switches on the ribosome turned on and off by thiostrepton and micrococcin. Mol. Cell 30, 26–38 (2008).

  20. 20.

    et al. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR–Cas9 system. Nat. Biotechnol. 32, 819–821 (2014).

  21. 21.

    et al. Activity of clinically relevant antimalarial drugs on Plasmodium falciparum mature gametocytes in an ATP bioluminescence ‘transmission blocking’ assay. PLoS ONE 7, e35019 (2012).

  22. 22.

    et al. Male and female Plasmodium falciparum mature gametocytes show different responses to antimalarial drugs. Antimicrob. Agents Chemother. 57, 3268–3274 (2013).

  23. 23.

    , & Developmental regulation of stage-specific ribosome populations in Plasmodium. Nature 342, 438–440 (1989).

  24. 24.

    , , , & Plasmodium vivax and Plasmodium falciparum ex vivo susceptibility to anti-malarials and gene characterization in Rondonia, west Amazon, Brazil. Malar. J. 13, 73 (2014).

  25. 25.

    & The in vitro and in vivo effects of mefloquine on Trypanosoma brucei brucei. J. Hyg. Epidemiol. Microbiol. Immunol. 36, 191–199 (1992).

  26. 26.

    , , , & In vitro effects of artemisinin ether, cycloguanil hydrochloride (alone and in combination with sulfadiazine), quinine sulfate, mefloquine, primaquine phosphate, trifluoperazine hydrochloride, and verapamil on toxoplasma gondii. Antimicrob. Agents Chemother. 38, 1392–1396 (1994).

  27. 27.

    , , & Structure of the human 80S ribosome. Nature 520, 640–645 (2015).

  28. 28.

    et al. Structure of the large ribosomal subunit from human mitochondria. Science 346, 718–722 (2014).

  29. 29.

    et al. Structure–activity relationships amongst 4-position quinoline methanol antimalarials that inhibit the growth of drug sensitive and resistant strains of Plasmodium falciparum. Bioorg. Med. Chem. Lett. 20, 1347–1351 (2010).

  30. 30.

    et al. Anti-malarial activity of a non-piperidine library of next-generation quinoline methanols. Malar. J. 9, 51 (2010).

  31. 31.

    , , , & Identification of resistance of Plasmodium falciparum to artesunate–mefloquine combination in an area along the Thai–Myanmar border: integration of clinico-parasitological response, systemic drug exposure, and in vitro parasite sensitivity. Malar. J. 12, 263 (2013).

  32. 32.

    et al. Structural basis for the inhibition of the eukaryotic ribosome. Nature 513, 517–522 (2014).

  33. 33.

    . et al. Dynamical features of the Plasmodium falciparum ribosome during translation. Nucleic Acids Res. 43, 10515–10524 (2015).

  34. 34.

    et al. Identification of Plasmodium falciparum specific translation inhibitors from the MMV malaria Box using a high throughput in vitro translation screen. Malar. J. 15, 173 (2016).

  35. 35.

    & Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

  36. 36.

    RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

  37. 37.

    , , & Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife 2, e00461 (2013).

  38. 38.

    Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife 3, e03665 (2014).

  39. 39.

    et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).

  40. 40.

    & Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

  41. 41.

    , & Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

  42. 42.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

  43. 43.

    et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011).

  44. 44.

    et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014).

  45. 45.

    , & Low-resolution refinement tools in REFMAC5. Acta Crystallogr. D 68, 404–417 (2012).

  46. 46.

    , , , & Correcting pervasive errors in RNA crystallography through enumerative structure prediction. Nat. Methods 10, 74–76 (2013).

  47. 47.

    et al. Molprobity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

  48. 48.

    et al. Reticulocyte-binding protein homologue 5—an essential adhesin involved in invasion of human erythrocytes by Plasmodium falciparum. Int. J. Parasitol. 39, 371–380 (2009).

  49. 49.

    et al. Isolation of viable Plasmodium falciparum merozoites to define erythrocyte invasion events and advance vaccine and drug development. Proc. Natl Acad. Sci. USA 107, 14378–14383 (2010).

  50. 50.

    et al. 4-quinolinemethanol derivatives as purine receptor antagonists (II) US patent 6,608,085 (2003).

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Acknowledgements

The authors thank I. Lucet, J. Boddey, S. Herrmann, G. McFadden, J. Rayner, A. Ruecker, M. Delves, H. Baumann, G. Murshudov and P. Emsley for discussions and experimental assistance, S. Chen and C. Savva for help with microscopy, and J. Grimmett and T. Darling for help with computing. The experimental data were made possible by Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. The research was directly supported by a National Health and Medical Research Council of Australia (NHMRC) Project Grant (APP1024678 to J.B. and W.W.), the Australian Cancer Research Foundation, a Human Frontier Science Program (HFSP) Young Investigator Program Grant (RGY0071/2011, to J.B.) and grants from the UK Medical Research Council (MC_UPA0251013, to S.H.W.S.). W.W. is an Early Career Development Awardee (APP1053801) from the NHMRC and was in receipt of a travel award from OzEMalaR to visit MRC–LMB UK to conduct experiments. X.-C.B. is supported by an EU FP7 Marie Curie Postdoctoral Fellowship. A.B. and I.S.F. are supported by grants to V. Ramakrishnan from the Wellcome Trust (WT096570) and the UK Medical Research council (MC_U105184332). J.B. was supported by a Future Fellowship (FT100100112) from the Australian Research Council (ARC) and is currently supported by an Investigator Award from the Wellcome Trust (100993/Z/13/Z). Additional support for this work came from a Pathfinder Award from the Wellcome Trust (105686).

Author information

Author notes

    • Wilson Wong
    • , Xiao-Chen Bai
    • , Brad E. Sleebs
    •  & Tony Triglia

    These authors contributed equally to this work.

Affiliations

  1. Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia

    • Wilson Wong
    • , Brad E. Sleebs
    • , Tony Triglia
    • , Jennifer K. Thompson
    • , Danushka S. Marapana
    • , Alan F. Cowman
    •  & Jake Baum
  2. MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK

    • Xiao-Chen Bai
    • , Alan Brown
    • , Israel S. Fernandez
    •  & Sjors H. W. Scheres
  3. Bio21 Molecular Science and Biotechnology Institute, Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia

    • Katherine E. Jackson
    • , Eric Hanssen
    •  & Stuart A. Ralph
  4. Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia

    • Alan F. Cowman
    •  & Jake Baum
  5. Department of Life Sciences, Imperial College London, South Kensington, London SW7 2AZ, UK

    • Jake Baum

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Contributions

W.W., X.-C.B., B.E.S., K.E.J., T.T., D.S.M., S.A.R., S.H.W.S. and J.B. designed all experiments. W.W., X.-C.B., B.E.S., K.E.J., A.B., T.T., D.S.M., J.K.T., E.H. and I.S.F. performed experiments. W.W., X.-C.B., B.E.S., K.E.J., T.T., A.B., J.K.T., S.A.R., A.F.C., S.H.W.S. and J.B. contributed to manuscript preparation.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Sjors H. W. Scheres or Jake Baum.

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DOI

https://doi.org/10.1038/nmicrobiol.2017.31

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