Transition State Mimetics of the Plasmodium Export Element Are Potent Inhibitors of Plasmepsin V from P. falciparum and P. vivax

Following erythrocyte invasion, malaria parasites export a catalogue of remodeling proteins into the infected cell that enable parasite development in the human host. Export is dependent on the activity of the aspartyl protease, plasmepsin V (PMV), which cleaves proteins within the Plasmodium export element (PEXEL; RxL↓xE/Q/D) in the parasite’s endoplasmic reticulum. Here, we generated transition state mimetics of the native PEXEL substrate that potently inhibit PMV isolated from Plasmodium falciparum and Plasmodium vivax. Through optimization, we identified that the activity of the mimetics was completely dependent on the presence of P₁ Leu and P₃ Arg. Treatment of P. falciparum-infected erythrocytes with a set of optimized mimetics impaired PEXEL processing and killed the parasites. The striking effect of the compounds provides a clearer understanding of the accessibility of the PMV active site and reaffirms the enzyme as an attractive target for the design of future antimalarials

PMV conservation and expression.

<p>(A) Structure and size of PMVHA proteins used in this study. Catalytic dyad residues DTG/DSG are shown including Asp to Ala mutations* in red. TM, transmembrane domain. (B) Immunoblot of infected erythrocytes with α-HA antibodies shows expression of PMVHA proteins in <i>P. falciparum</i>. Sizes indicate that the signal peptides were removed (PfPMVHA, 69.1 kDa; PvPMVHA, 60.9 kDa). (C) Immunoblotting of infected erythrocytes with rabbit α-PfPMV antibodies (Rα-PfPMV) validates they are specific for PfPMV. Endogenous PfPMV is the lower band (lanes 1, 3, 4, 5), and the larger band corresponds to 3× HA-tagged PfPMV (lanes 2, 4). Aldolase is a loading control in (B) and (C) and shows slight overloading of some lanes compared to others. (D, Top) Immunofluorescence micrographs show rabbit α-PfPMV antibodies (Rα-PfPMV, green) label PfPMV in the ER. Colocalizations were performed with mouse α-PfPMV antibodies (Mα-PfPMV, red), shown previously to label PMV in the ER <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-Klemba1" target="_blank">[16]</a>. (Middle) α-HA antibodies (red) label PfPMVHA (Top) and PvPMVHA (Bottom) in the parasite ER. (Bottom) α-HA antibodies (red) label PvPMVHA in the ER, as shown by clocalization with ERC (green). (E) Immunopurified PfPMVHA and PvPMVHA cleave KAHRP peptides containing the PEXEL sequence RTLAQ but not peptides containing point mutations R>K, L>I, or RL>A. Pf and Pv PMVmutHA proteins with catalytic D>A mutations did not cleave the KAHRP RTLAQ peptide. (F) Overexpression of PfPMVmutHA from episomes in <i>P. falciparum</i> 3D7 impairs growth relative to expression of a similar episomal construct encoding a mini PfEMP1HA reporter (miniVarHA). Parasites expressing episomes were selected on 5 nM WR99210 (WR). Two starting inocula were used in triplicate wells, and parasitaemia was determined at 72 h. *<i>p</i><.0001 (<i>t</i> test). Data are mean ± SEM from duplicate experiments.</p

WEHI-916 is lethal to <i>P. falciparum</i> 3D7.

<p>(A) Dose-response curves of <i>P. falciparum</i> 3D7 in the presence of 916, 024, or 025. EC₅₀ values are shown. (B) Parasitemia measured at 72 h (<i>y</i>-axis) following drug treatment at rings (30 min postinvasion) and replacement of the medium with inhibitor-free medium (wash-out) at the time intervals shown (<i>x</i>-axis). (C) Parasitemia at 72 h (<i>y</i>-axis) after replacement of inhibitor-free medium with media containing compounds at the intervals shown (<i>x</i>-axis). Parasitemia was determined by FACS in (A–C) and is relative to DMSO treatment in (B) and (C). Concentrations are as follows: 916, 024, 025 (15 µM); CQ, chloroquine (150 ng/ml); ART, artemisinin (100 ng/ml). Error bars in (A–C) are mean ±SEM from duplicate experiments. (D) Light micrographs of Giemsa-stained parasites 16 and 32 h after drug treatment at early rings (15 µM). 916-treated parasites failed to develop into trophozoites and did not recover. Ring parasites treated with E-64 (10 µM) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-terKuile1" target="_blank">[22]</a> contained swollen food vacuoles (arrow) due to inhibition of proteases involved in hemoglobin degradation; however, treatment with DMSO, 916, 024, or 025 did not cause swelling. Swelling was quantified using 500 infected cells per condition in duplicate. Scale bar is 6 µm.</p

PMV knockdown or overexpression modulates sensitivity to WEHI-916.

<p>(A) Immunoblot with Rα-PfPMV antibodies shows successful integration of the PMVHA-<i>glmS</i> or -M9 plasmid (M9 is an inactive <i>glmS</i> riboswitch control). The upper band (α-PfPMV blot) in lane 1 (denoted by *) is nonspecific. The same blot is shown below, probed with α-HA antibodies. HSP70 is a loading control. (B) Knockdown of PMV in <i>P. falciparum</i> NF54 following 5 mM GlcN treatment. (Left) 0 h GlcN treatment of trophozoites causes no knockdown. (Center) The 24 h GlcN treatment of trophozoites causes ∼80% knockdown of PMV in subsequent rings compared to “−GlcN.” (Right) 48 h GlcN treatment of trophozoites causes ∼90% knockdown of PMV in subsequent trophozoites compared to “−GlcN.” A small degree of knockdown is seen for M9, indicating GlcN has a minor effect. (C) PMV knockdown by GlcN has no significant effect on parasite growth rate (<i>p</i> = .6250). Trophozoites were treated with 0 mM or 5 mM GlcN and parasitaemia determined 48 h later by flow cytometry. Data are % growth “+GlcN” relative to “−GlcN,” and data are mean ±SEM of a representative of duplicate experiments. (D) PEXEL processing of PfEMP3-GFP in <i>P. falciparum</i> parasites expressing PMVHA-<i>glmS</i> is reduced more by 916 treatment when PMV is knocked down [+GlcN (5 mM for 48 h prior to addition of 916)]. Densitometry shows the ratio of uncleaved to PEXEL-cleaved protein in each lane beneath the blot. Note that PfEMP3-GFP expression is lower in “+GlcN” parasites despite relatively similar HSP70 levels. (E) Dose-response curves of <i>P. falciparum</i> expressing PMVHA-<i>glmS</i> shows parasites have enhanced sensitivity to 916 following PMV knockdown (3.3-fold decrease in EC₅₀). Parasitemia was determined 72 h after addition of 916 to ring parasites with or without PMV knockdown (knockdown ring parasites were obtained by adding 6 mM GlcN to trophozoites for 24 h). GlcN and 916 were maintained in the culture medium throughout. (F) Dose-response curves of <i>P. falciparum</i> overexpressing PvPMVHA or a mini PfEMP1HA reporter (miniVarHA) in the presence of 5 nM WR99210 show parasites have increased resistance to 916 when PMV is overexpressed (1.9-fold increase in EC₅₀).</p