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.

There is an urgent need to develop new strategies and agents to treat malaria because of the emergence of resistance against antimalarial therapies in clinical use. , This is particularly significant given that malaria continues to pose a substantial global health burden, with an estimated 263 million cases and 597,000 deaths reported in 2023.

Plasmodium falciparum expresses 10 cathepsin-D-like aspartyl proteases termed plasmepsins (PM). PMI-IV are located in the digestive vacuole and are involved in the initial phase of hemoglobin digestion with some functional redundancies. − PMVI-VIII, are expressed in the transmission stage of the lifecycle and have no essential role in the asexual stage. ,− PMV, PMIX, and PMX are all essential in the asexual blood stage of infection; PMIX and PMX are required for parasite egress and invasion of red blood cells (RBCs) and also play a critical role in other stages of the lifecycle. − Highlighting the druggability of these plasmepsins, − inhibitors against PMIX/X are currently in clinical trials (NCT06294912). PMV is essential for protein export and parasite development in both the asexual and sexual stages of the lifecycle − and therefore also remains an attractive multistage antimalarial target.

During the asexual blood stage, parasites export a large number of proteins to the host RBC, which is essential for nutrient and waste exchange, sustenance, and to evade the host immune system. Most of these exported proteins contain a pentameric N-terminal motif called the Plasmodium export element (PEXEL). − The PEXEL is essential for transportation of proteins from the parasite endoplasmic reticulum (ER), through the parasitophorous vacuole membrane via the Plasmodium translocon of exported proteins (PTEX) into the host RBC. , The PEXEL is comprised of a highly conserved motif among all Plasmodium species and is designated by the consensus sequence, RxLxE/Q/D. The PEXEL motif is recognized and cleaved on the C-terminal side of Leu by the ER resident aspartyl protease, PMV. , The remaining C-terminal xE/Q/D stub is then N-acetylated by a putative acetylase committing the protein for export to the host RBC. ,

One approach to develop a small molecule inhibitor of PMV is to mimic the native PEXEL motif. Historically, aspartyl proteases have been targeted with transition state mimetics that mimic the catalytic intermediate in the proteolysis of the substrate. Examples include peptidomimetics targeting HIV protease, renin, and BACE1. − The earliest example of a PEXEL transition state peptidomimetic was WEHI-916 (Figure ). Through genetic knockdown and overexpression studies of PMV in parasites, we demonstrated that WEHI-916 is a potent inhibitor of PMV and modestly inhibits the export of PEXEL containing proteins and parasite development. , By replacing the P3 Arg in WEHI-916 with canavanine (Cav), an analogue named WEHI-842 was subsequently produced and has a 10-fold improvement in both affinity for PMV and efficacy against P. falciparum compared to WEHI-916 (Figure ). , Further optimization of the peptidomimetic compounds at the P₂ position generated two analogues named WEHI-600 and WEHI-601 (Figure ). Both WEHI-600 and WEHI-601 show a 4-fold improvement in both PMV inhibition and parasite efficacy.

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.

The peptidomimetic inhibitors have been robust chemical probes in the pharmacological validation of PMV. A confounding factor in the development of these inhibitors is the disparity between the biochemical inhibition of PMV and the concentrations of these peptidomimetics required to block the PEXEL cleavage and protein export and kill the parasite. Here, we investigated these PMV peptidomimetic inhibitors through chemical and genetic techniques and confirmed their on-target activity against Plasmodium falciparum. The observed disparity is likely due to cell permeability and the abundance of PMV expression in the parasite, requiring heightened concentrations of the peptidomimetic to completely nullify its proteolytic function.

Results

Plasmepsin and Human Biochemical Selectivity

Given that the structural architecture of parasite plasmepsins is similar, it is possible that the peptidomimetics designed to target PMV were also binding other essential plasmepsin proteins, PMIX and PMX, and in turn inhibiting parasite development through one or multiple targets. At the time of the PMV peptidomimetic development, recombinant PMIX and PMX were not accessible. , More recently, reliable sources of recombinant PMIX and PMX have become available, , allowing us to assess the biochemical activity of the PMV peptidomimetics WEHI-601, WEHI-916, and WEHI-842 against these proteins. These assays indicated that the peptidomimetics had no activity (IC₅₀ > 50 μM) against PMIX, while some modest activity against PMX was observed with WEHI-916 and WEHI-842 (IC₅₀ 0.99 and IC₅₀ 1.85 μM, respectively) but not WEHI-601 (IC₅₀ > 99 μM) (Table ). Despite this activity against PMX, it is notable that WEHI-916 and WEHI-842 were found to inhibit PMV 11- to 62-fold more potently than PMX (Table ).

1. Biochemical Activity of Peptidomimetics against Asexual Stage Essential Plasmepsins and Human Aspartyl Proteases .

compound	parasite EC50 (SD)	PMV IC50 (SD)	PMIX IC50 (SD)	PMX IC50 (SD)	cathepsin D IC50 (SD)	BACE1 IC50 (SD)	renin IC50 (SD)
WEHI-916	6.05 (1.79)	0.09 (0.01)	50.75 (25.58)	0.99 (0.02)	ND	ND	ND
WEHI-842	1.07 (0.17)	0.03 (0.03)	>99.00	1.85 (0.24)	0.52* (0.61)	>1.00	>1.00
WEHI-601	0.70 (0.17)	0.005 (0.002)	>99.00	>99.00	ND	>11.10*	ND

^aAll values are in μM, with values derived from at least three biological replicates except for those indicated with a *, which represent two biological replicates. Parasite EC₅₀ values are derived from the 3D7 parasite strain. ND indicates not determined.

To assess the peptidomimetic inhibitor’s selectivity, we then evaluated the panel of compounds against human aspartyl proteases. This indicated that the peptidomimetic WEHI-842 does not inhibit the human aspartyl proteases BACE1 and renin (IC₅₀ > 1.00 μM) (Table ). WEHI-601 also did not display any activity against BACE1 (IC₅₀ > 11.0 μM). WEHI-842, however, demonstrated modest activity against human cathepsin D (IC₅₀ 0.52 μM). Despite this activity, WEHI-842 has a 17-fold greater activity against PMV (IC₅₀ 0.03 μM), indicating that selectivity toward the parasite aspartyl protease PMV was maintained.

Target Engagement Studies Reveal Plasmepsin V as the Primary Target of WEHI-601

Next, we investigated the on-target activity of the peptidomimetics against PMV within the parasite. WEHI-601 was selected to carry out these experiments as it displayed the highest potency against both recombinant PMV and P. falciparum growth viability. First, we used the principle of solvent-induced protein precipitation (SIP), which reveals protein–ligand interactions based on the alteration of protein susceptibility to denaturation after ligand binding in the presence of an organic solvent. − We performed SIP assays whereby schizont lysate obtained from a hemeagglutinin (HA)-tagged PMX parasite line was treated with 5 μM of WEHI-601, or vehicle solvent, DMSO. We then challenged the lysate with a mixture of 0–25% (v/v) organic solvent acetone/ethanol/formic acid (AEF) and the soluble proteins were isolated by centrifugation. The soluble fractions were separated by Western blot and probed with the anti-PMV antibody (Figure A). Here, from a concentration of >17% AEF, we observed significantly more soluble PMV in the WEHI-601-treated group when compared to the DMSO control, indicating target engagement between the peptidomimetic and PMV.

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).

We next attempted to investigate any possible engagement between WEHI-601 and PMX in these SIP assays using anti-HA to detect PMX. Despite multiple attempts, we were unable to observe any reproducible pattern of PMX detection via solvent denaturation (Figure S1). This was possibly due to PMX not being compatible with the organic solvent method via Western blotting. Thus, to further investigate the selectivity of the peptidomimetic inhibitors in parasites, we conducted a live cell thermal profiling assay with 3D7 parasites coupled with global proteomics analysis. Following a proteome integral solubility alteration (PISA) experimental format, we separated the samples after thermal challenge and pooled samples into low and high denaturing groups (gradient 50–60 °C and gradient 62–72 °C). Through data independent acquisition mass spectrometry (DIA-MS) analysis, the relative protein abundance was determined. This assay revealed 18 proteins that were either significantly stabilized or destabilized in the presence of WEHI-601, compared to the DMSO control (Table S1). Among these hits, PMV was found to be the most significantly stabilized protein (Figure B, p < 0.00001). Other proteins that were significantly stabilized included the putative serpentine receptor SR1 (PF3D7_1131100), an ATPase subunit ClpY (PF3D7_0907400), and schizont egress antigen-1, SEA1 (PF3D7_1021800) (Figure B). We also investigated other detectable proteins in the plasmepsin family and found that only PMIV exhibited slight stabilization in response to WEHI-601 treatment (P < 0.01), while no other plasmepsin proteins showed any stabilizing effect (Table S1, Figure S2). Taken together, both the SIP and thermal-PISA experiments demonstrated that the peptidomimetic inhibitor WEHI-601 targets and is highly selective of PMV.

PMV Peptidomimetics Do Not Alter the Parasite Metabolome

We next wanted to determine whether changes in the parasite metabolome could be detected upon treatment with WEHI-601. Here, we treated parasites with either WEHI-601, the inactive compound WEHI-024, or DMSO for two durations: a short 5 h treatment at 22–24 h post invasion (hpi) or longer 16 h treatment at 6–8 hpi. In both treatment durations, the overall metabolomes of WEHI-601 and control-treated parasites were unchanged, except for some short chain peptides that were significantly increased (Figure ). In the 5 h treatment, some peptides identified to be perturbed following treatment with WEHI-601 were predicted to be derived from hemoglobin (Table S2). These peptides have previously been identified in metabolomic studies of inhibitors of hemoglobin catabolism. Notably, however, most of these peptides also exhibited a significant upward trend in response to the inactive control treatment (Table S2), indicating that they are off-target and are unrelated to the antiparasitic activity. In the 16 h treatment, a similar trend was observed, with the same peptides increasing in both WEHI-601 and the inactive WEHI-024 treatment. The only significantly increased peptide in the WEHI-601 treatment that was not found in WEHI-024-treated samples was Ala–Cys–Pro–Ser (Table S2).

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.

Overall, the metabolomic study showed that the peptidomimetic PMV inhibitor WEHI-601 did not significantly affect the metabolome of P. falciparum 3D7 parasites compared to the structurally similar PMV inactive control compound WEHI-024.

WEHI-601-Resistant Parasites Contain a Mutation in Plasmepsin v

To consolidate the on-target activity of the PMV peptidomimetic inhibitors, we performed a drug pressure resistance study. To generate resistance, we treated 3D7 parasites with WEHI-601 at 0.9 μM until parasite death was observed by Giemsa-stained blood smears. The compound was then removed, and parasites were allowed to recover. This drug cycling was repeated for 3 cycles to select for resistant parasites. WEHI-601 was shown to have a 3-fold difference against the parental line (EC₅₀ 0.38 μM) compared to 3D7 (EC₅₀ 0.11 μM) using an LDH assay (Figure S3). Five clonal parasite strains were generated from the 3E parental strain. WEHI-601 was shown to have similar activity against each of the five clonal strains, indicating that the resistance obtained was stable (Figure S3).

Three of the five resistant clones (F10, D6, and B11) were selected for whole genome sequencing. Upon subsequent retesting of the three clonal lines, it was found that clone F10 had a greater shift in EC₅₀ against WEHI-601 compared to the other two clones (Figure A). Analysis of the WEHI-601-resistant genomes revealed one shared single nucleotide variant (SNV) across all three clones. This was a Thr 371 Pro mutation in the plasmepsin v gene (PF3D7_1323500) (Table ). Resistant clone F10 contained additional nonsynonymous SNVs in three further genes including in a vacuolar protein sorting-associated protein 53 (PF3D7_0727000), tRNA methyltransferase (PF3D7_1019800), and a cell cycle-associated protein (PF3D7_1220300) (Table ). No additional variants across the resistant clones were detected.

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.

2. Nonsynonymous Single Nucleotide Variants (SNVs) Found in WEHI-601-Resistant Clones F10, D6, and B11 .

clone	chromosome	position	ref	Alt	Gene_ID	gene_description	codon change
F10	Pf3D7_13_v3	976513	A	C	PF3D7_1323500	plasmepsin V	T371P
D6	Pf3D7 13_v3	976513	A	C	PF3D7_1323500	plasmepsin V	T371P
B11	Pf3D7_13_v3	976513	A	C	PF3D7_1323500	plasmepsin V	T371P
F10	Pf3D7_07_v3	1148030	G	A	PF3D7_0727000	vacuolar protein sorting-associated protein 53	P1010H
F10	Pf3D7_10_v3	805275	C	A	PF3D7_1019800	tRNA methyltransferase	E770K
F10	Pf3D7_12_v3	808470	G	A	PF3D7_1220300	cell cycle-associated protein	R259K

^aHighlighted rows indicate an SNV that was shared among the resistant clones.

The PMV^T371P mutation was then mapped to the X-ray structure of Plasmodium vivax PMV in complex with WEHI-601, demonstrating that the mutation was localized adjacent to the S₂ pocket of PMV, which accommodates the P₂ CyHex group of WEHI-601 (Figure B). This result indicates that the mutation may be induced by the unnatural CyHexGly amino acid in the P₂ position of WEHI-601.

Examining the location of this SNV across multiple Plasmodium spp. including P. falciparum, P. vivax, Plasmodium knowlesi, Plasmodium malariae, Plasmodium yoelii, Plasmodium cynomolgi, and Plasmodium berghei showed that the site of the mutation and surrounding amino acids are highly conserved (Figure S4). This could indicate that this region plays an important role in the PEXEL substrate specificity across Plasmodium species.

Since this region in PMV could be important for the enzyme activity, we determined if the mutation led to a fitness cost in parasite growth. To assess this, we grew the clones F10, B11, and D6 containing the PMV^T371P, in addition to a wild-type 3D7 control, for three cycles of growth. Each cycle, we took samples and conducted an LDH assay as a proxy for parasite growth (Figure S5A). No significant difference was observed in the amplification rate between WEHI-601-resistant clones and 3D7 (p > 0.05) between cycles two and three (Figure S5B). This indicates that the T371P mutation in PMV does not induce any growth defects in the parasites.

Evaluation of PMV Peptidomimetic Inhibitors with Varied P₂ Side Chain against WEHI-601-Resistant Parasites

Since no fitness cost was observed in the WEHI-601-resistant parasites, we hypothesized that the mutation would not affect the binding of natural P₂ amino acids that are commonly found in PEXEL substrates, such as Val, to the S₂ pocket of PMV. To assess this, we tested if other PMV peptidomimetic inhibitors with smaller P₂ side chain groups were cross-resistant to the WEHI-601-resistant parasites. These included WEHI-842 and WEHI-916, both of which have a Val in the P₂ position, WEHI-404 with a NorVal in the P₂ position (or n-propyl group as the P₂ side chain), and WEHI-912 with Ile as the P₂ residue (Figure ). To evaluate parasite growth upon treatment, P. falciparum 3D7 parasites were treated with these PMV peptidomimetic inhibitors in a dose–response for 72 h and subsequent parasitemia was measured using an LDH assay (Figure S6). This revealed that WEHI-916, WEHI-842, and WEHI-404 had equipotent activity against the WEHI-601-resistant clones F10, D6, and B11 compared to 3D7 (Table ). WEHI-912, containing an Ile in the P₂ residue, was found to display a minimal shift in EC₅₀ against the resistant clones of approximately 1.3-fold (Table ). This result supports that the T371P mutation in PMV effects binding of the larger unnatural CyHex group, and to a smaller extent Ile, but does not significantly affect the binding of Val or NorVal, both of which contain smaller side-chain groups.

3. Activity of PMV Peptidomimetic Inhibitors with Different P₂ Side Chains against WEHI-601-Resistant Parasites in LDH Growth Assays .

EC50 μM (SD) fold-change	WEHI-601	WEHI-842	WEHI-916	WEHI-404	WEHI-912
3D7	0.31 (0.07)	0.69 (0.15)	7.44 (1.75)	3.04 (0.36)	0.42 (0.04)
F10	0.78 (0.10)	0.81 (0.09)	7.41 (0.10)	2.19 (0.46)	0.55 (0.11)
	2.5-fold	1.2-fold	1.0-fold	0.7-fold	1.3-fold
B11	0.43 (0.04)	0.72 (0.10)	4.50 (3.14)	3.15 (0.43)	0.60 (0.12)
	1.4-fold	1.0-fold	0.6-fold	1.0-fold	1.4-fold
D6	0.43 (0.05)	0.57 (0.09)	4.64 (0.74)	2.61 (0.20)	0.55 (0.11)
	1.4-fold	0.8-fold	0.6-fold	0.9-fold	1.3-fold

^aEC₅₀ values present an average of >2 independent experiments against wild-type 3D7 parasites and WEHI-601-resistant clones F10, B11, and D6, which all contain a T371P mutation in PMV. The fold-resistance for each clone is listed, which is calculated by the fold-change in EC₅₀ relative to 3D7.

PMV^T371P Mediates Resistance to WEHI-601

To investigate the effect of PMV^T371P in mediating resistance to WEHI-601, we employed reverse genetics using CRISPR-Cas9 to engineer the SNV into wild-type parasites. Here, we designed a donor plasmid consisting of endogenous wild-type plasmepsin v homology regions (“HR1 and HR2”) with HR1 fused to a recodonized region that contained either the wild-type PMV^T371 or the PMV^T371P mutation. For expression and localization analysis, we included a hemagglutinin (HA) tag and a human dihydrofolate reductase expression cassette (hDHFR) to select for transfectants resistant to WR99210. A gRNA was designed binding to a protospacer adjacent motif (PAM) upstream of the SNV to mediate the Cas-9 cleavage, which was then repaired through homologous recombination using donor plasmid as the template (Figure A).

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.

After transfectant parasites of both PMV^T371-HA and PMV^T371P-HA were successfully obtained, genomic DNA was extracted, and integration PCRs were performed using primers located in both the 5′UTR and HR1 (“a/b”) and 5′UTR and HA tag (“a/c”) regions (Figure A). This revealed that only transfected parasites with the donor plasmid could obtain a product with the PCR “a/c”, indicating that integration had successfully occurred (Figure B). These PCR products were sequenced and showed that the desired wild-type PMV^T371 (ACC) or PMV^T371P (CCG) sequences were present (Figure C). HA-tagged protein expression was confirmed by an anti-HA immunoblot (Figure D).

We next investigated PMV^T371-HA and PMV^T371P-HA parasites against WEHI-601 via a 72 h LDH growth assay. Here, we included 3D7 as the parental control in addition to the original F10 clone. It was observed that there was a 3.1-fold increase in EC₅₀ in the PMV^T371P-HA parasite line when compared to the equivalent wild-type control line PMV^T371-HA (EC₅₀ 823 vs 266 nM) (Figure E). The fold change in growth observed was comparable to that seen in the 3D7 wild-type line when compared to the F10 clone (EC₅₀ 345 vs 657 nM) (Figure E). This result indicates that the T371P mutation in PMV mediates resistance toward WEHI-601.

Discussion

Due to the prevalence of resistance to clinically used antimalarials in malaria endemic regions, the need for novel antimalarial discovery and development is urgent. PMV provides an attractive target due to its essentiality in multiple stages of the Plasmodium lifecycle. , Our efforts have focused on developing peptidomimetic compounds that mimic the natural structure of PEXEL proteins to block the PMV function. However, discourse has remained regarding the on-target activity of these peptidomimetic inhibitors, so herein we confirmed their activity against PMV using multiple strategies.

In this study, by testing PMV peptidomimetic inhibitors WEHI-601, WEHI-842, and WEHI-916 against recombinant P falciparum PMV, PMIX, and PMX, we found the inhibitors presented good selectivity against PMV with low-mid nanomolar affinity. They did not display activity against PMIX but some modest affinity against PMX was observed with WEHI-842 and WEHI-916. This may be due to the structural similarity between PMV and PMX, whereby both proteins share a similar aspartic protease core structure. Despite this modest activity against PMX, the selectivity index of the peptidomimetic inhibitors against PMV remains high, indicating that PMV inhibition remains their main mode of action against the parasite. Intriguingly, the IC₅₀ of the inhibitors against recombinant PMV did not align with the EC₅₀ values against parasite growth. This could possibly be due to the inherent peptide-like characteristics of the peptidomimetics having limited cell membrane permeability, resulting in modest parasite activity. Further work is required to enhance the potency of these peptidomimetic inhibitors against the parasite in vitro. Importantly, however, the PMV peptidomimetic inhibitors displayed little to no activity against the human aspartyl proteases BACE1 and renin, which is advantageous for their drug development potential.

Target-compound engagement methods SIP and thermal PISA profiling both demonstrated stabilization of PMV by WEHI-601 in both in vitro and live cell conditions, respectively. The second most significantly stabilized protein by WEHI-601 was SR1. In a previous study, immunoprecipitation experiments of a PMV-HA parasite line enriched SR1, indicating that SR1 is likely an interactor of PMV. Since we have now shown that WEHI-601 stabilizes PMV, it is probable that through its interaction with PMV, SR1 would also be stabilized in the target engagement assays. This finding underscores the value of live-cell target engagement studies in revealing physiologically relevant pathways and interactors. ClpY (PF3D7_0907400) was another protein hit that displayed significant stabilization in the intact-cell thermal PISA profiling. ClpY is an ATPase subunit of a mitochondria ATP-dependent protease, which has been shown to be important in programmed cell death in the P. falciparum asexual stage development. SEA1 or schizont egress antigen-1 (PF3D7_1021800) was also significantly stabilized by WEHI-601, which is known to act as an upstream regulator for packaging of nuclei during parasite schizogony. In the absence of strong stabilization, and since both ClpY and SEA1 are not involved in the protein trafficking pathway, these proteins are unlikely to be stabilized with direct interactions with WEHI-601.

Live cell thermal-PISA detected all 7 of the plasmepsins that are expressed in the asexual blood stage (PMI-V, IX-X). Apart from PMV, WEHI-601 failed to stabilize any other plasmepsin proteins except for some minimal stabilization of PMIV. This may have resulted from the sequence similarity in the catalytic center of PMV and PMIV. This affinity, however, is unlikely to contribute to the mechanism of action of WEHI-601 since we have not observed any phenotype associated with hemoglobin digestion and PMIV is not essential for asexual stage development. ,

To complement the proteomics studies, we generated WEHI-601-resistant parasites, and whole genome sequencing revealed a T371P mutation in PMV. We found that this mutation site in PMV is adjacent to the H372 position, which forms a hydrogen bond to S368 that binds within the PfPMV catalytic aspartate D365. This implies that the region surrounding the T371P mutation site is critical to the PMV function, which may explain why the region surrounding the mutation is highly conserved across several different Plasmodium spp. We did not, however, observe any fitness cost to parasites that contained the PMV^T371P. However, this was performed only across three cycles of growth; so extending this out to evaluate if there may be a long-term growth fitness cost would be desirable. Nonetheless, it is possible that PMV^T371P only impairs the access of the large cyclo-hexane P₂ residue of WEHI-601 to access the S₂ pocket of PMV but the smaller natural amino acid P₂ residues of endogenous substrate PEXEL proteins remain unaffected. Supporting this, we found no cross resistance from parasites containing PMV^T371P to compounds containing P₂ Val (WEHI-842 and WEHI-916), P₂ NorVal (WEHI-404), and P₂ Ile (WEHI-912).

To further investigate the role of the T371P mutation in WEHI-601 resistance, we introduced the PMV^T371P mutation into wild-type 3D7 parasites, which demonstrated this mutation mediated resistance against WEHI-601. In the original resistant clone F10, however, we also identified three additional genes with SNVs including in the vacuolar protein sorting-associated protein 53 gene, which encodes the putative protein PfVPS53. In yeast and mammalian systems, VPS53 is a subunit of the Golgi-associated retrograde protein (GARP) complex, known to facilitate the fusion of endosome-derived transport carriers with the trans-Golgi network. − In Plasmodium, three of the four GARP subunits are expressed, indicating it is likely to have a similar function in the parasite. Given that PfVPS53 may play a role in protein trafficking within the secretory pathway, it could represent a secondary mutation contributing to the mechanism of action of the peptidomimetic inhibitors. Therefore, further efforts to reverse engineer this SNV would be worthwhile. Of note, different methods for whole genome sequencing F10 and the other two clones B11 and D6 were utilized; MinIon Nanopore sequencing for clone F10; and Illumina next-generation sequencing (NextSeq) for clones B11 and D6. This could be a contributing factor to differences found in their analyses.

We also investigated whether WEHI-601 would alter the parasite metabolome through a metabolomics analysis. We found no significantly distinguishable changes in metabolites between the WEHI-601-treated group and the control treatments, indicating that the PMV peptidomimetic inhibitors do not have a significant effect on parasite metabolome. This phenomenon has been observed with other compounds that inhibit export, such as KAF156, an antimalarial agent with an unknown target in the secretory pathway, which similarly showed no effect on the parasite’s metabolome.

Conclusion

In this study, through proteomics and genetic methods, we demonstrated the on-target activity of PMV peptidomimetic inhibitors to provide a platform for the further development of such inhibitors. However, a conditional knockdown approach has shown that parasite survival can be maintained with less than 1% of normal PMV levels, suggesting that PMV activity must be reduced to extremely low levels to significantly impair parasite growth. Thus, PMV peptidomimetic inhibitors require further chemical modification to improve potency and permeability before they achieve a sufficiently high efficacy to inhibit parasite growth in vitro at concentrations in line with current antimalarial compounds in the clinic. Nonetheless, continued efforts to identify more drug-like PMV inhibitors remain worthwhile, as has been demonstrated previously. The peptidomimetic compounds and PMV mutant lines described here provide valuable tools to probe the PMV function and druggability. While the current peptidomimetics inhibitors are not suitable for antimalarial development, they establish a foundation for future optimization toward more drug-like candidates. Due to PMV’s highly conserved sequence in Plasmodium spp. and essential function during other stages of the lifecycle, there is potential for PMV inhibitors to evolve as a cross-species and multistage antimalarial.

Experimental Section

Parasite Culture and Lines Used in This Study

P. falciparum parasites were cultured as previously reported in human O-type RBCs (Australian Red Cross Blood Bank) at 4% hematocrit in RPMI-HEPES media supplemented with 5% v/v heat-inactivated human serum (Australian Red Cross Blood Bank) and 5% v/v albumax (Gibco) (herein referred to as complete RPMI) unless specified. P. falciparum wild-type parasites in all experiments were laboratory strain 3D7 parasites unless specified. A P. falciparum parasite line expressing hemeagglutinin-tagged Plasmepsin X (PMX-HA) was used for solvent-induced protein precipitation experiments.

Compounds

WEHI-601, WEHI-912, WEHI-404 (referred to as compounds 27, 29, and 10 in ref ), WEHI-842,, and WEHI-916 were synthesized as reported before. The above compounds were all dissolved in DMSO to a 10 mM stock solution and stored at −20 °C.

Plasmepsin Biochemical Assays

This was performed as per with the following changes. Starting concentrations of compounds were as follows: 10–100 μM (PvPMV assays), 1–11 μM (renin, cathepsin D and BACE-1 assays), or 100 μM (PfPMX and PfPMIX assays).

LDH Malstat Growth Assay

These assays were performed as previously described. Synchronized ring-staged parasites were treated with serial diluted compounds at 0.5% parasitemia with 2% hematocrit in a 100 μL final volume in 96-well plates. A nine-point titration was performed with a 1 in 2 serial dilution in complete RPMI media with starting concentrations of WEHI-601 (4.8 μM), WEHI-912 (5 μM), WEHI-404 (20 μM), WEHI-842 (20 μM), and WEHI-916 (20 μM). Plates were incubated at 37 °C for 72 h before parasitemia was quantified using an LDH malstat assay as previously described. Dose–response curves and EC₅₀s were analyzed through GraphPad Prism 10.3.0 using four-parameter log­(inhibitor) vs response nonlinear regressions.

WEHI-601-Resistant Parasite Generation

This was performed as previously described with a selection pressure of 0.9 μM, which equated to approximately 10 × EC₅₀.

Fitness Cost Parasite Growth Assay

WEHI-601-resistant clones F10, B11, and D6, and a 3D7 parasite line were synchronized via 5% sorbitol lysis 48 h before the assay setup. For the assay setup, ring-stage parasites were adjusted to 0.5% parasitemia and 2% hematocrit and incubated at 37 °C. Samples were then collected each cycle for 3 cycles and stored frozen until assay completion. To prevent parasite overgrowth, cultures were diluted 1:8 at each cycle with this dilution factored into the analysis. Parasite growth was quantified by using the LDH assay described above.

Whole Genome Sequencing of Clone F10 WEHI-601-Resistant Parasites

This was performed as previously described with alterations outlined below. The PMV isolate and 3D7 control isolate were sequenced in one sequencing run and performed on a MinION platform with MIN106/R9.4.1 flow cells and MinIT (Software 18.09.1) to generate fast5 files that were then basecalled using (Guppy V3.1.5 + 781ed57) and demultiplexed using Porechop (V0.2.4). Reads were aligned against the P. falciparum 3D7 reference genome (PlasmoDB version 31) using minimap2 (V2.1.7) using the map-ont preset. Candidate SNVs in subtelomeric, centromeric, or hypervariable regions were removed using bedtools subtract (V2.26.0) to only retain SNVs in the core genome. To examine structural variants, demultiplexed fastq files were additionally aligned using a sensitive long read aligner NGMLR V0.2.7 (https://github.com/philres/ngmlr) and again processed with samtools utilities (V1.7) to sort and index the alignment files. The aligned files were then examined for structural variants using Sniffles V1.0.11 (https://github.com/fritzsedlazeck/Sniffles) and filtered to contain high read support ≥10 and filtered based on the structural variant length ≥200bp. SNVs in 12 genes that were common to both the F10 WEHI-601-resistant genome and an unrelated resistant genome sequenced concurrently were excluded from the analysis. The data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB82704.

Whole Genome Sequencing of Clones D6 and B11 WEHI-601-Resistant Parasites

This was performed as previously described with the following changes: Copy number analysis was performed using the R package QDNaseq v1.28.0 with 1 kbp windows. Copy number windows were filtered to exclude regions that were centromeric, telomeric, or had mappability of less than 0.5 based on 30mers generated by GenMap. The data for this study have been deposited in the ENA at EMBL-EBI under accession number PRJEB82800.

Genetically Engineered T371P PMV Mutant P. falciparum Parasites

To introduce the WEHI-601-resistant mutation into 3D7 wild-type parasites, 533 bp of endogenous plasmepsin v (PF3D7_1323500) was synthesized as homology region 1 (“HR1”) along with the recodonized following endogenous sequence including either the WT or T371P mutation with a 3× hemeagglutinin tag at the 3′ end. The entire “HR1-pmv-HA” fragment was synthesized (GenScript), and Gibson assembly (Gibson Assembly Master Mix, New England Biolabs) was performed to insert this fragment into parasite vector p1.2. Primers for Gibson assembly were designed through the NEBuilder online program https://nebuilder.neb.com/#!/ (listed in Table S3).

The guide RNA sequences (stated in Table S3) were designed through the online program https://chopchop.cbu.uib.no with a PAM (Table S3). The gDNA fragment was fused into the pUF1-Cas9G plasmid, which included the Cas-9 gene (In-Fusion HD cloning kit, Takara).

The donor plasmid was linearized and transfected with the guide plasmid into late schizont-stage 3D7 parasites (Amaxa Basic Parasite Nucleofector Kit 2, Lonza) as described in. Parasites that had integrated the donor plasmid containing a human dihydrofolate resistance (hDHFR) gene into their chromosome were selected with 2.5 nM WR99210. Once viable parasites were obtained, genomic DNA was extracted, and integration was confirmed via PCR using primers listed in Table S3. These PCR products were then Sanger sequenced (Australian Genome Research Facility) to confirm the presence of the desired mutation or wild-type sequences.

Solvent-Induced Precipitation Protein Profiling of Plasmepsin V

Lysate of the PfPMX-HA parasite line was prepared as described in without the addition of protease inhibitors. Briefly, parasite lysate was treated with either WEHI-601 (10 μM) or DMSO (0.1% v/v) for 3 min before being aliquoted into a 0–25% acetone/ethanol/formic acid mixture (AEF) at a 50:50:1 (v/v) solution. After agitation at 800 rpm for 20 min at 37 °C, the precipitated protein was removed by centrifugation at 17,000g for 20 min at 4 °C and the supernatant was transferred into 1× NuPAGE LDS Sample Buffer (Invitrogen) with 1:100 2-mercaptoethanol (Sigma-Aldrich). The sample was boiled for 3 min, and proteins were separated on 4–12% acrylamide gels (NuPAGE, Invitrogen). Blots were then probed with anti-PMV antibody (1:1000), anti-HA (1:1000) (Roche), and anti-PfSTART1 (1:1000) with corresponding HRP antibodies (1:2000) (antirabbit-HRP, Merck; antirat-HRP, Roche).

Intact-Cell Thermal PISA Profiling with WEHI-601

DIA Thermal PISA (Proteome Integral Solubility Alteration) Assay

The experiment was carried out in 4 biological replicates. Synchronized mature-stage 3D7 parasites (35–41 hpi) were exposed to WEHI-601 or the DMSO [Sigma] vehicle control for 1 h at standard culture conditions. Parasites were pelleted through centrifugation and resuspended in WEHI-601/DMSO-supplemented PBS, each split into 13 identical aliquots, and transferred onto a 96-well plate. Samples were heated in a PCR machine [Biorad] for 3.5 min to different temperatures across 50–72 °C gradient (at 2 °C intervals) or to 37 °C as a nondenaturing control. Cells were lysed by 3× flash freeze/thawing using liquid N₂, followed by 10× mechanical sheering with a 29-gauge needle-syringe, and denatured protein removal through filtration at 0.2 μM level. The soluble phase was recovered and pulled together in equivolume ratios into two samples; gradient 1: 50–60 °C and gradient 2: 62–72 °C, respectively. Sample preparation for proteomic analysis was carried out using the modified SP4 protocol. In brief, 20 μg of protein was reduced [20 mM TCEP, 100 mM TEAB] for 10 min at 95 °C and alkylated with 55 mM CAA for 30 min. Following addition of 20 μL of PureCube Carboxy magnetic beads [Cube biotech] and neat ice-cold ACN to a final concentration of 80%, samples were incubated on a thermomixer for 20 min at RT at 800 rpm and pelleted down at 3000g for 5 min. Beads were washed 3× with 80% ethanol and following SN removal dried in a SpeedVac. Dried beads were subjected to sequential digestion with LysC (3 h, 1:50 enzyme/protein ratio) and trypsin (overnight, 1:50 enzyme/protein ratio). The resulting digest was acidified with TFA to a final 1% concentration and desalted on T3 C18 stage tips [Affinisep] according to manufacturer’s instructions.

MS Data Acquisition and Data Analysis

Following resuspension in 0.1% formic acid and 2% ACN, peptide samples were loaded on to a C18 fused silica column (inner diameter 75 μm, OD360 × 15 cm length, 1.6 μm C18 beads) packed into an emitter tip [IonOptics] and separated on a 25 min analytical gradient on a Neo Vanquish liquid chromatography system [Thermo Scientific] interfaced with MS [Orbitrap Astral Mass Spectrometer, Thermo Scientific] and analyzed in a DIA mode. Peptide identification was carried out in DIA-NN 1.8.1 using in silico spectral library generated from Uniprot P. falciparum (UP000001450) and human (UP000005640) reference proteomes. Two missed cleavages and 2 variable modifications [ox­(M) and Ac­(N-term)] were allowed. Differential abundance data analysis (moderated t-test) of P. falciparum proteins was conducted in the R environment using precursor normalized MaxLFQ data for proteins detected with ≥2 peptides. Hit selection criteria included p value <0.01, >0.2 log2-fold change in protein abundance, and protein detection across all samples in the comparison. Raw Mass Spectrometry data for DIA Thermal PISA experiments is available at JPOSTrepo, a member of the ProteomeXchange Consortium under the following accession numbers: JPST004118 and PXD069420.

Metabolomics

Cell Culture and Drug Incubations for LC–MS Metabolomics Analysis

3D7 parasites were grown in RPMI medium containing hypoxanthine and 0.5% (wt/vol) Albumax (Gibco). For the 5 h treatment, parasites were synchronized by double 5% (wt/vol) sorbitol lysis, 14 h apart and cultured for a further 52 h to ensure the experiment was performed on early trophozoite stage cultures ∼22–24 hpi. Parasites were adjusted to 6% parasitemia and treated with WEHI-601 or WEHI-024 (0.9 μM) for 5 h. Following the treatment duration, parasites were harvested using magnet separation to achieve enriched trophozoite samples (>80% parasitemia). For the 16 h treatment, parasites were tightly synchronized to 0 ± 2 h by magnet harvesting late-stage segmented schizonts, adding the magnet elute to uninfected red blood cells (RBCs) and performing sorbitol lysis 2 h later. Parasites were cultured for a further 6 h (∼6–8 hpi) before treatment with WEHI-601 or WEHI-024 (0.9 μM) for 16 h. Both experiments included untreated controls that contained equivalent amounts of dimethyl sulfoxide (DMSO; as a vehicle).

Sample Extraction and LC–MS Metabolomics Analysis

After drug treatments, metabolites of infected RBCs (iRBCs) were collected as previously described, with minor modifications. Metabolites of iRBCs in the 5 and 16 h treatments were extracted in either 100 or 200 μL of methanol, respectively.

Metabolite analysis was performed by liquid chromatography–mass spectrometry LC–MS using hydrophilic interaction liquid chromatography (HILIC) and high-resolution (Q-Exactive Orbitrap, Thermo Fisher) MS as previously described. , Briefly, samples (10 μL) were injected onto a Dionex RSLC U3000 LC system (Thermo) fitted with a ZIC-pHILIC column (5 μm particle size, 4.6 mm × 150 mm; Merck) and 20 mM ammonium carbonate (A) and acetonitrile (B) were used as the mobile phases. A 30 min gradient starting from 80% B to 50% B over 15 min, followed by washing at 5% B for 3 min and re-equilibration at 80% B, was used. MS was used with a heated electrospray source operating in positive and negative modes (rapid switching) and a mass resolution of 35,000 from m/z 85 to 1275. Sample injections within the experiment were randomized to avoid any impact of systematic instrument drift on metabolite signals. Retention times for ∼350 authentic standards were checked manually to aid in metabolite identification.

LC–MS Metabolomics Data Processing

Metabolomics data sets were analyzed using IDEOM. Raw files were converted to mzXML with msconvert; extraction of LC–MS peak signals was conducted with the Centwave algorithm in XCMS, alignment of samples, and filtering of artifacts with mzMatch, and additional data filtering and metabolite identification were performed in IDEOM (Supporting Information file S1). In the 5 and 16 h-treatments, 411 and 453 putative metabolites were identified, respectively. Metabolite abundance was determined by the LC–MS peak height and was normalized to the average for untreated samples. Statistical analyses used Welch’s t-test (p-value< 0.05) and Pearson’s correlation (Microsoft Excel). Principal-component analysis (PCA) was generated in Metaboanalyst, a web interface. This study is available at the NIH Common Fund’s National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org under the following study IDs: ST004290, ST004291.

Glossary

Abbreviations

AEF

acetone/ethanol/formic acid

DIA-MS

data independent acquisition mass spectrometry

ER

endoplasmic reticulum

GARP

Golgi-associated retrograde protein

HA

hemeagglutinin

hDHFR

human dihydrofolate reductase

HPI

hours post invasion

HR

homology region

LDH

lactate dehydrogenase

PEXEL

Plasmodium export element

PISA

proteome integral solubility alteration

PM

plasmepsin

PMV

plasmepsin V

PTEX

Plasmodium translocon of exported proteins

RBCs

red blood cells

SIP

solvent-induced protein precipitation

SNV

single nucleotide variant.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.5c00742.

• Replicates blots of SIP assays (Figure S1); plasmepsins in thermal proteome profiling assay (Figure S2); clonal resistance to WEHI-601 (Figure S3); Plasmodium species sequence alignment of the PMV T371 region (Figure S4); fitness cost of PMV­(T371P) in parasites (Figure S5); dose–response curves of PMV­(T371P) parasites against P₂ residues (Figure S6); and uncropped blots used in this study (Figure S7); untargeted metabolomics analysis of peptides (Table S2); and sequences and primers used to construct donor plasmids (Table S3) (PDF)

• Hits from intact thermal PISA profiling (Table S1) (XLSX)

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.