a–c, Substrate cleavage profile of activated human andP. falciparum 20S proteasome.a, Activation of the human andP. 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 theP. 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’st-test).d,e, Inhibition potencies of the vinyl sulfone inhibitors.d, Table of IC₅₀ values for each inhibitor in 1-h treatedP. 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 inFig. 1f andExtended Data Fig. 2b were quantified to calculate the IC₅₀ values (for gel source data and replicates, seeSupplementary 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 ofP. falciparum at ring stage or non-confluent HFFs. Data are mean ± s.d.;n = 6 parasite cultures from two independent experiment of triplicates forP. 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, wheren = 6 cell cultures from two independent experiments of triplicates.

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, seeSupplementary Fig. 1b).c, HFF orP. 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 inP. falciparum is derived fromFig. 4c at the indicated concentrations to allow for direct comparison with other compounds (for gel source data, seeSupplementary Fig. 1e).

a–e, Evaluation of the single-particle analysis of theP. 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 theP. 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.f–h, 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 theP. 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.

a, WLW-vs was incubated in early trophozoiteP. 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, seeSupplementary 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 theP. 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 × 10⁶P. 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.

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 inP. falciparum. Error bars, s.d. (n = 6 parasite cultures for WLL-vs from triplicates of two independent experiments, andn = 8 parasite cultures for WL(d)L-vs over three independent experiments).c, PurifiedP. 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, seeSupplementary Fig. 1g).d, A mixed-stage culture ofP. 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, seeSupplementary Fig. 1h.e, Geimsa stain of 1 h treatedP. falciparum ring 24 h after inhibitor was added. Scale bar, 600 μm.

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)