Fig 1.
The P. falciparum-selective peptide vinyl sulfone proteasome inhibitors WLL and WLW are highly potent against schizonts and early ring-stage parasites irrespective of their K13 status.
(A and B) Mean ± SEM IC50 values for (A) WLL or (B) WLW, tested in 72 hr dose-response assays with asynchronous parasites. These inhibitors were assayed against Cam3.II or V1/S parasites expressing either WT K13 or the ART-resistant R539T or C580Y variants. Assays were conducted on three to nine independent occasions in duplicate. Mann-Whitney U tests showed no significant differences between K13 WT lines and their isogenic variants on either background. IC50 values and statistics are reported in S2 Table. (C and D) Mean ± SEM IC50 values for (C) WLL or (D) WLW, tested in 3 hr exposures followed by washes to remove drug and a further 69 hr of culture in drug-free medium. Parasitemias were measured by flow cytometry. Assays were initiated with either early rings (0–3 hr post-invasion) or trophozoites (tested 24 hr after early rings). Assays were performed with Cam3.II or V1/S lines expressing K13 WT or C580Y, on three to four independent occasions in duplicate. Mann-Whitney U tests showed no significant differences between K13 WT lines and their isogenic variants. Two-way ANOVA tests comparing Cam3.II and V1/S parasite lines revealed no significant differences for WLL, and a small but significant difference for WLW. **P value = 0.005. IC50 values and statistics are reported in S3 Table. (E-G) Tightly synchronized Cam3.II parasites expressing K13 WT or C580Y were treated for 1 hr with (E) 150 nM WLL, (F) 2000 nM WLW, or (G) 150 nM DHA at five intervals, beginning with distinct stages. Drug was removed by washing and cultures were continued for a further 71 hr in drug-free medium prior to measuring parasitemias. Data are shown as mean ± SEM percent survival for drug-treated parasites, calculated relative to DMSO vehicle-treated cultures tested in parallel. Assays were performed on three (WLL and WLW) or two (DHA) independent occasions in duplicate. Percent survival values are reported in S4 Table.
Table 1.
In vitro resistance selections.
Table 2.
Mutations identified by whole-genome sequencing of WLL- or WLW-pressured parasite lines.
Fig 2.
WLL- and WLW-pressured mutant parasite lines display modest gains of resistance.
(A and B) Mean ± SEM IC50 values are shown for the Cam3.II or V1/S lines expressing (blue) WT or (red) C580Y K13, exposed to (A) WLL or (B) WLW for 72 hr. Assays were performed on five to seven independent occasions in duplicate. Shown above each bar is the compound used for resistance selections (thin hatching, WLL-selected; thick hatching, WLW-selected). Mutant lines (see Table 2 and S7 Table) are illustrated beside their parental drug-sensitive line. Statistical significance was examined using Mann-Whitney U tests. **P value <0.01; ns, not significant. IC50 and IC90 values and associated statistics are reported in S8 Table and S9 Table, respectively.
Fig 3.
In silico modeling of 20S β2, β5 and β6 mutations reveals disruption of inhibitor binding.
(A-E) Modeling of WLL binding. (A) Structure of the WLL peptide-based inhibitor, indicating positions P1-P3 and the electrophilic vinyl sulfone warhead that reacts with the β5 subunit catalytic threonine residue (Thr1). (B-E) Docking of WLL into the β5 active site. The β5 and β6 subunits are shown in light blue and dark blue, respectively. Arrows highlight the P3 tryptophan residue of the ligand. (B) Docking of WLL into the WT β5 site of the cryo-EM derived P. falciparum 20S proteasome model. (C-E) Docking of WLL into the β5 active site of molecular dynamics-simulated models with the WLL-selected mutations, namely (C) β5 A20S (D) β6 A117V and (E) β6 S208L, demonstrating their potential impact on WLL binding. (F-J) Modeling of WLW binding. (F) Structure of the WLW peptide-based inhibitor, indicating positions P1-P3 and the electrophilic vinyl sulfone warhead that reacts with the β2 subunit catalytic threonine residue (Thr1). (G-J) Docking of WLW into the β2 active site. The β2 and β3 subunits are shown in orange and green, respectively. Arrows highlight the P1 tryptophan residue of the ligand. (G) Docking of WLW into the WT β2 site of the cryo-EM derived P. falciparum 20S proteasome model. (H-J) Docking of WLW into the β2 active site of the superposed WLW-selected mutation models, i.e. (H) β2 C31F (I) β2 C31Y and (J) β2 A49E, and their potential impact on WLW binding.
Fig 4.
Activity-based probe profiling of 20S β2 and β5 active sites permits quantification of the impact of 20S subunit mutations on inhibitor binding.
(A) P. falciparum schizont lysates treated for 1 hr with WLL or WLW at concentrations ranging from 0.5 to 50 μM, or mock-treated with DMSO, then incubated for 2 hr with the BMV037 probe. Samples were run on 12% SDS-PAGE gels. Data show results from one representative experiment. (B-D) Bar charts showing the concentrations at which WLL or WLW achieved half-maximal inhibition of probe labeling to each subunit. Data are shown as mean ± SEM IC50 values from two independent experiments (of which one is shown in panel A). (B) Inhibition of β2 subunit by WLL; (C) Inhibition of β5 subunit by WLL (D); inhibition of β2 subunit by WLW. IC50 values are reported in S10 Table.
Fig 5.
WLW synergizes with multiple classes of structurally and functionally diverse antimalarials.
Isobolograms of WLW and (in descending order) DHA, OZ439, MB, b-AP15 and ESI tested on asynchronous parasites and highly synchronized rings and trophozoites. Cam3.II K13WT or Cam3.II K13C580Y parasites were exposed to compounds mixed at fixed ratios of their individual IC50 values (1:0, 4:1, 2:1, 1:1, 1:2, 1:4, 0:1). Asynchronous parasites were exposed for 72 hr and parasitemias were determined by flow cytometry. Highly synchronized rings (0–3 hr post-invasion) or trophozoites (tested 24 hr later) were exposed for 3 hr, followed by drug washouts and continued culture for 69 hr in drug-free media. Fractional IC50 (FIC50) values were plotted for each drug combination and fixed ratio and results were compared against a hypothetical isobole line illustrating a perfectly additive interaction (dashed line). Data show results of two independent isobologram assays (shown in different shades), each performed in duplicate, tested against Cam3.II K13WT (blue) and Cam3.II K13C580Y (red). Synergy is evidenced by individual FIC50 pairwise values falling below the dashed line of additive interactions (FIC50 = 1). DHA, dihydroartemisinin; ESI, eeyarestatin I; MB, methylene blue.
Fig 6.
WLL and WLW show differential interactions with distinct classes of antimalarials, including synergy with DHA and the related ozonide OZ439, and antagonism with CQ and PPQ.
(A-C) Heat maps of interactions between the WLL or WLW proteasome inhibitors and distinct antimalarial agents. Assays used the Cam3.II K13WT and Cam3.II K13C580Y lines. Parasites were exposed to compounds mixed at fixed ratios of their individual IC50 values (1:0, 4:1, 2:1, 1:1, 1:2, 1:4, 0:1). (A) Asynchronous parasites were exposed for 72 hr and parasitemias were determined by flow cytometry. (B) Highly synchronized rings (0–3 hr post-invasion) or (C) trophozoites (tested 24 hr later) were exposed for 3 hr, followed by drug washouts and continued culture for 69 hr in drug-free media. Values represent the mean of the sums of the FIC50 values over the five fixed ratios of the two test compounds (excluding the 1:0 and 0:1 points). Assays were conducted on two to four independent occasions in duplicate. Data for WLW and the five top compounds (DHA, OZ439, MB, b-AP15 and ESI) are presented as isobolograms in Fig 5. ATQ, atovaquone; CQ, chloroquine; DHA, dihydroartemisinin; ESI, eeyarestatin I; LMF, lumefantrine; MB, methylene blue; PPQ, piperaquine. Means of the sums of FIC50 (mean ΣFIC50) values are reported in S12 Table.