Letter | Published:

Structure- and function-based design of Plasmodium-selective proteasome inhibitors

Nature volume 530, pages 233236 (11 February 2016) | Download Citation

Abstract

The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation1. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle2,3,4,5. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents2,6. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the β2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials7,8, we observe growth inhibition synergism with low doses of this β2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next-generation anti-malarial agents.

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Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The cryo-EM map and the atomic coordinates of the inhibitor-bound Plasmodium 20S proteasome have been deposited in the Electron Microscopy Data Bank (EMDB) and PDB under accession numbers EMD-3231 and 5FMG, respectively.

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Acknowledgements

This work was support by National Institutes of Health grants R01AI078947, R01EB05011 to M.B., and by the Medical Research Council grant MC-UP-1201/5 to P.C.A.dF. H.L. was supported by an NSS-PhD scholarshpip from the Agency for Science, Technology and Research (A*STAR) Singapore. W.A.v.d.L. was supported by a Rubicon fellowship from the Netherlands Organization for Scientific Research (NWO). A.J.O. and C.S.C. were supported by the Program for Breakthrough Biomedical Research (PBBR) and the Sandler Foundation. I.T.F. was supported by American Heart Association grant 14POST20280004. We acknowledge support from the Australian Research Council and the Australian National Health and Medical Research Council. We thank K. Chotivanich for providing PL2 and PL7 parasites. We thank E. Yeh’s group for help with P. falciparum D10 culture and for use of their equipment. We thank J. Boothroyd for providing the human fibroblast cells. We thank E. Morris and R. Henderson for discussions on image processing, FEI fellows and C. Savva for assisting in the use of the Titan Krios microscope, S. Chen for EM support, and J. Grimmet and T. Darling for computing support.

Author information

Author notes

    • Hao Li
    •  & Anthony J. O’Donoghue

    Present addresses: Molecular Engineering Laboratory, Biomedical Sciences Institute, Agency for Science, Technology and Research, Singapore 138673 (H.L.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093, USA (A.J.O.).

Affiliations

  1. Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Hao Li
    •  & Wouter A. van der Linden
  2. Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Hao Li
    • , Euna Yoo
    • , Ian T. Foe
    •  & Matthew Bogyo
  3. Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94158, USA

    • Anthony J. O’Donoghue
    •  & Charles S. Craik
  4. Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, Melbourne 3010, Victoria, Australia.

    • Stanley C. Xie
    •  & Leann Tilley
  5. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK

    • Paula C. A. da Fonseca

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Contributions

H.L., A.J.O., L.T., C.S.C., P.C.A.dF. and M.B. designed the experiments and wrote the manuscript. H.L., W.L. and E.Y. performed chemical synthesis and analysis. H.L., S.C.X. and I.T.F. performed the inhibitor studies. P.C.A.dF. did the electron microscopy and image analysis. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Paula C. A. da Fonseca or Matthew Bogyo.

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    Supplementary Figures

    This file contains Supplementary Figure 1, showing the raw data for Figures 1f, 4c and Extended Data Figures 2b, 2c, 4a, 5c, 5d.

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https://doi.org/10.1038/nature16936

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