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


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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Figure 1: Substrate profile of the activated human and P. falciparum 20S proteasome guides inhibitor design.
Figure 2: Structure of the P. falciparum 20S proteasome core bound to the inhibitor WLW-vs, determined by cryo-EM and single-particle analysis.
Figure 3: Structural comparison of the P. falciparum and human proteasome 20S core active sites.
Figure 4: Exploiting differences in the β2-subunits of the two proteasome species for selective parasite killing.

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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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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.

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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.

Corresponding authors

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

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Activity, substrate specificity and inhibition of human and P. falciparum 20S proteasome.

ac, Substrate cleavage profile of activated human and P. falciparum 20S proteasome. a, Activation of the human and P. falciparum 20S proteasome by human PA28α. Activity was determined by cleavage of the fluorogenic substrate Suc-LLVY-amc. Error bars, s.d. n = 3 purified proteasome in technical replicates. b, c, iceLogos of cleavage sequences that are uniquely processed either by the P. falciparum (b) or by the human proteasome (c). Amino acids that are most and least favoured at each position are shown above and below the axis, respectively. Lower-case ‘n’, norleucine; amino acids in black text are statistically significant (P < 0.05, unpaired two-tailed Student’s t-test). d, e, Inhibition potencies of the vinyl sulfone inhibitors. d, Table of IC50 values for each inhibitor in 1-h treated P. falciparum and human 20S proteasome. IC50 values are determined from three independent experiments of inhibitor pretreatment followed by activity labelling of the 20S proteasome (n = 3 purified proteasome). Gels in Fig. 1f and Extended Data Fig. 2b were quantified to calculate the IC50 values (for gel source data and replicates, see Supplementary Fig. 1a, b). Data are mean ± s.d. e, Table of EC50 values for each of the inhibitors in 1 h and 72 h treatment of P. falciparum at ring stage or non-confluent HFFs. Data are mean ± s.d.; n = 6 parasite cultures from two independent experiment of triplicates for P. falciparum treatments; n = 9 cell cultures from three independent experiments of triplicates for HFF treatment, except for 1 h WLW-vs, 1 h LLW-vs, and 72 h LLL-vs, where n = 6 cell cultures from two independent experiments of triplicates.

Extended Data Figure 2 Proteasome inhibitors preferentially inhibit β2 of the P. falciparum proteasome.

a, Vinyl sulfone inhibitors are synthesized from the Boc-protected amino acid by first generating the Weinreb amide, followed by the Horner–Wadsworth–Emmons reaction and standard peptide coupling. b, Purified human 20S proteasome was pre-treated for 1 h at 37 °C with each inhibitor followed by addition of activity-based probe MV151 (ref. 40) to assess for human proteasome activities (for gel source data, see Supplementary Fig. 1b). c, HFF or P. falciparum culture was treated for 1 h at 37 °C with each inhibitor, followed by compound washout and post-lysis activity-based probe labelling. Gel shown for WLL-vs in P. falciparum is derived from Fig. 4c at the indicated concentrations to allow for direct comparison with other compounds (for gel source data, see Supplementary Fig. 1e).

Extended Data Figure 3 Evaluation of the final cryo-EM map and molecular model of the P. falciparum 20S proteasome core.

ae, Evaluation of the single-particle analysis of the P. falciparum 20S proteasome core bound to the inhibitor WLW-vs. a, Cryo-EM image of the sample analysed, with molecular images of side views of the complex (normal to its long axis) indicated by rings. The image greyscale was inverted to show the protein densities in white. b, Individual sections of the 3D map, as determined by the 3D reconstruction algorithm (without further sharpening, masking, or Fourier filtering), are represented as grey scale. These sections are 1 Å thick and reveal the quality of the reconstruction, as the protein densities are clearly resolved against a very smooth background, with regions showing the pattern of α-helices (box) and the clear separation of sheet-forming β-strands (arrows) indicated. c, Evaluation of the model of the P. falciparum 20S proteasome core using MolProbity39. d, Resolution estimate of the cryo-EM map by Fourier shell correlation. The curves correspond to the correlation obtained against the protein model (red) and the correlation between maps determined from two halves of the data (blue). The resolution was estimated from the curve against the model where the 0.5 correlation coefficient criterion41 yields an estimate of 3.6 Å. The correlation coefficient can be seen to fall to a local minimum at ~6 Å and then recover at higher resolutions for both Fourier shell correlation curves. This behaviour is consistent with the rotationally averaged amplitude spectra of both the cryo-EM map and the coordinates (e). This region of the amplitude spectra contains reduced structural information, typical of protein scattering, indicating that these effects in the Fourier shell correlation curves arise from a genuine local reduction in the signal:noise ratio. fh, Accessibility of the human 20S proteasome active sites to the inhibitor WLW-vs, using the protein model of the human proteasome core complex bound to an LLL-vs inhibitor20. Protein coordinates of the human proteasome 20S core (PDB 5A0Q) β2 (f), β1 (g), and β5 (h) active sites were aligned to the coordinates of the P. falciparum proteasome β2-subunit bound to the WLW-vs inhibitor. The model of the human 20S proteasome active sites is represented as van der Waals surfaces with the superimposed WLW-vs inhibitor shown as sticks.

Extended Data Figure 4 Intact cell treatment and in vivo treatment of vinyl sulfone inhibitors.

a, WLW-vs was incubated in early trophozoite P. falciparum culture for 3 h, washed out, and the parasite lysate was incubated with probe BMV037. Top gel, the fluorescent scan; bottom gel, the silver stain. For gel source data, see Supplementary Fig. 1f. b, Body weight of WLL-vs- and vehicle-treated Balb/c mice after compound treatment by tail vein injection (Fig. 4e), expressed as a percentage of the original body weight on day 3 before compound treatment. Body weight of vehicle-treated mice decreased after day 6 of infection as part of the response to the natural resolution of the P. chabaudi infection. Treatment day is indicated by arrow; n = 6 mice for each group; error bars, s.d. e, Balb/c female mice infected with 1 × 106 P. chabaudi parasites from passage host on day 0 were treated with a single bolus dose of vehicle (45% polyethylene glycol (relative molecular mass 400), 35% propylene glycol, 10% ethanol, 10% DMSO, and 10% (w/v) 2-hydroxyproyl-β-cyclodextrin; n = 4 mice) or WLL-vs at 40 mg kg−1 (n = 5 mice), 60 mg kg−1 (n = 4 mice), and 80 mg kg−1 (n = 3 mice) formulated in the vehicle. Treatment was performed on day 2 after infection as indicated by the arrow and administered by intraperitoneal injection. Parasitaemia was monitored daily by Giemsa stain of thin blood smears. Error bars, s.d.

Extended Data Figure 5 Assessing off-target activities of WLL-vs.

a, Structures of WLL-vs and its diastereomer WL(d)L-vs. b, Dose–response curves of WLL-vs and WL(d)L-vs after 72 h treatment in P. falciparum. Error bars, s.d. (n = 6 parasite cultures for WLL-vs from triplicates of two independent experiments, and n = 8 parasite cultures for WL(d)L-vs over three independent experiments). c, Purified P. falciparum 20S proteasome was treated for 1 h at 37 °C with 10 μM of WLL-vs and WL(d)L-vs (left) or a range of concentrations of WL(d)L-vs. Residual activity was assessed by probe BMV037 (for gel source data, see Supplementary Fig. 1g). d, A mixed-stage culture of P. falciparum was treated for 1 h with WLL-vs at 37 °C, followed by BODIPY-TMR-DCG04 for a further 1 h. Samples were directly loaded onto SDS–polyacrylamide gel electrophoresis for analysis. JPM-OEt (100 μM) was included as positive control. The fluorescent scan is shown at the top and the Coomassie stain is shown at the bottom. For gel source data, see Supplementary Fig. 1h. e, Geimsa stain of 1 h treated P. falciparum ring 24 h after inhibitor was added. Scale bar, 600 μm.

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This file contains Supplementary Figure 1, showing the raw data for Figures 1f, 4c and Extended Data Figures 2b, 2c, 4a, 5c, 5d. (PDF 1629 kb)

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Li, H., O’Donoghue, A., van der Linden, W. et al. Structure- and function-based design of Plasmodium-selective proteasome inhibitors. Nature 530, 233–236 (2016).

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