Deconvolution of the On-Target Activity of Plasmepsin V Peptidomimetics in Plasmodium falciparum Parasites

Abstract

Plasmepsin V (PMV), an essential aspartyl protease, plays a critical role during the asexual blood stage of infection of Plasmodium by enabling the export of parasite proteins into the host red blood cell. This export is vital for parasite survival and pathogenesis, making PMV an attractive target for antimalarial drug development. Peptidomimetic inhibitors designed to mimic the natural substrate of PMV have demonstrated potent parasite-killing activity by blocking protein export. While these compounds have been instrumental in validating PMV as a bona fide antimalarial target, inconsistencies between their biochemical potency and cellular activity have raised questions regarding their precise mechanism of action. In this study, we employed chemoproteomic approaches, including solvent-induced protein precipitation and intact-cell thermal profiling, to demonstrate PMV target engagement by the peptidomimetics. To further support these findings, we generated parasite lines exhibiting reduced sensitivity to peptidomimetics. Through whole-genome sequencing of these parasite lines, a single nucleotide variant within the pmv gene was revealed. This mutation was later validated using reverse genetics, confirming its role in mediating resistance. Together, these data provide strong evidence that the peptidomimetics exert their antimalarial activity by directly targeting PMV. These findings further support the potential of PMV as a validated and promising target for future antimalarial drug development.

Keywords: Plasmodium; antimalarial; aspartyl protease; malaria; plasmepsin.

1

Structure of PMV peptidomimetic inhibitors (A) WEHI-916, (B) WEHI-842, (C) WEHI-601, (D) WEHI-912, and (E) WEHI-404. The P₂ residue is highlighted in red.

2

WEHI-601 demonstrates target engagement for plasmepsin V. (A) Solvent-induced protein precipitation (SIP) assays. The parasite lysate was treated with DMSO (“–”) or 5 μM WEHI-601 (“+”). The lysate was challenged in acetone/ethanol/formic acid mixture (AEF, v/v/v = 50:50:1) gradient 0–25%. The soluble fraction of protein was extracted, and soluble proteins were separated out via Western blot and probed with anti-PMV antibody. PfSTART1 was probed with anti-PfSTART1 antibody as a loading control. Replicate blots can be found in Figure S1. (B) Live cell thermal proteome integral solubility alteration (PISA) profiling of WEHI-601 target engagement. Volcano plots depicted differential soluble protein abundance analysis (moderated t-test) for WEHI-601 (10 μM) and DMSO-treated parasites following heat pulse heat challenge (n = 4 biological replicates, mean ± SD). Nonsignificant (ns) proteins were plotted in gray. Destabilized are plotted in blue, and stabilized proteins are plotted in red. Hit selection cutoffs at 0.73 log2 fold change and p < 0.01 are indicated with dashed lines. The top four significantly stabilized hits are shown in the bar graphs representing the relative soluble protein abundance in WEHI-601- and DMSO-treated samples, across three thermal challenge conditions tested (x-axis). P-values can be found in Table S1. Bar graphs represent the average value of 4 biological replicates (±SD).

3

Untargeted metabolomics analysis of 3D7 parasites following treatment with WEHI-601, WEHI-024 (negative control), or DMSO control. Heat map profile of peak intensities of all metabolites from (A) a 5 h drug exposure of enriched parasites at 22–24 h post invasion (hpi) or (B) a 16 h drug exposure of parasites at 6–8 hpi. All compounds were incubated at a concentration of 0.9 μM; heat maps show two biological replicates with two technical replicates, except for DMSO, which had one technical replicate in the second biological replicate. Red, blue, and yellow indicate increase, decrease, or no change in the relative abundance of metabolites based on the relative peak intensity abundance, respectively. Principle component analysis (PCA), showing scores plot for components one and two for the 5 h treatment (C) and 16 h treatment (D). Data points indicate individual sample replicates within each treatment, and the shaded area denotes 95% confidence interval.

4

Generation of parasite resistance against WEHI-601 reveals a T371P mutation in plasmepsin V. (A) Three clonal resistance lines (F10, B11, and D6) were generated against WEHI-601, and EC₅₀’s were determined in parasite growth LDH assays. Error bars depict the standard deviation of 3 biological replicates. Statistical analyses via a one-way ANOVA comparing the mean of 3D7 EC₅₀’s vs the resistant clones. **** indicates p < 0.0001; ns indicates not significant. P-values for B11 and D6 compared to 3D7 were 0.11 and 0.10, respectively. Dose–response curves can be found in Figure S4A. (B) The structure of PvPMV in complex with WEHI-601 demonstrating the S₂ pocket within PMV in which the T371P mutation is located.

5

Introduction of T371P into plasmepsin v in 3D7 parasites mediates resistance to WEHI-601. (A) Design of the donor plasmid to introduce SNV (T371P) into 3D7 parasites. Homology regions (HRs) were designed to the 5′ flank (HR1) and 3′ flank (HR2) where recodonized plasmepsin v (pmv) followed after HR1 (shown in orange). Cas9 was directed by a synthetic RNA to the cleavage site to perform double crossover homologous recombination. Human dihydrofolate reductase (hDHFR) was introduced to allow transfected parasites selectable by WR92210. Primers were designed to confirm correct integration, where a was located in the 5′ untranslated region (UTR), while b was located in the HR1 region, and c was located within the hemeagglutinin (HA) tag. (B) PCRs using these primers were performed with genomic DNA from 3D7 and both WT and mutant CRISPR lines using the primers a, b, and c in (A) where the two products in the red box were sequenced. (C) Sanger sequencing confirmed the presence of the T371P (ACC–CCG) in PMV^T371P-HA parasite genomic DNA. (D) Western blot with anti-HA demonstrated that the transfected lines contained a HA tag with the expected size of 72 kDa. Anti-HSP70 antibody was a loading control. (E) Dose–response curves over a 72 h LDH assay shows that the activity of WEHI-601 is reduced against both original F10 clone and transfected PMV^T371P-HA parasite lines. Error bars are SD, which are indicated in brackets. EC₅₀ values indicate an average value of the three independent experiments.