Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle

Abstract

Artemisin combination therapy (ACT) is the main treatment option for malaria, which is caused by the intracellular parasite Plasmodium. However, increased resistance to ACT highlights the importance of finding new drugs. Recently, the aspartic proteases Plasmepsin IX and X (PMIX and PMX) were identified as promising drug targets. In this study, we describe dual inhibitors of PMIX and PMX, including WM382, that block multiple stages of the Plasmodium life cycle. We demonstrate that PMX is a master modulator of merozoite invasion and direct maturation of proteins required for invasion, parasite development, and egress. Oral administration of WM382 cured mice of P. berghei and prevented blood infection from the liver. In addition, WM382 was efficacious against P. falciparum asexual infection in humanized mice and prevented transmission to mosquitoes. Selection of resistant P. falciparum in vitro was not achievable. Together, these show that dual PMIX and PMX inhibitors are promising candidates for malaria treatment and prevention.

Keywords: Plasmodium; antimalarial; humanized mouse; malaria; merozoite; plasmepsin; plasmepsin IX; plasmepsin X.

Graphical abstract

Figure 1

Identification of Compounds that Inhibit P. falciparum (A) Screen using an aspartyl protease compound library to identify inhibitors of P. falciparum growth. (B) Chemical structure of compounds and EC₅₀. (C) WM5 and WM4 suppress P. berghei infection. Two independent experiments; n = 6, mean ± SD. ^∗∗p < 0.005, ^∗∗∗p < 0.0005. (D) Structure and EC₅₀ of R-WM382. (E) Growth curves for WM5 and 4 resistant P. falciparum. Experiments in triplicate, mean ± SEM. (F) Genome of P. falciparum WM5 and four resistant lines. Top: copy number (10 kb bins) of 3D7-WM4.2 compared to 3D7 for chromosomes, four replicates in four colors. Bottom: 30 kb region (1 kb bins). Gene numbers found at PlasmoDB: https://plasmodb.org/.

Figure 2

Enzyme Activity, Selectivity, and Substrate Specificity of rPMX (A) PMX-HA immuno-detection after pull-down with WM856. FT, flow through; E1, E2, E3, elution fractions. Left: unbound and eluted proteins from WM856-coupled beads. Center: with 0.2 μM WM382. Right: Uncross-linked beads. (B) IC₅₀ for WM382, WM4, and WM5 inhibition of PMX with Rh2N peptide. Mean ± SD. (C) K_i, for WM382,WM4, and WM5 with PMV and PMX, EC₅₀ for P. falciparum and HepG2 cells. Mean ± SD (three experiments). (D) rPMX activity for peptide cleavage. Mutant controls, mut; RFU, relative fluorescence units. Mean ± SD. (E) Rate of enzyme activity and K_m of rPMX for Rh2a/b and SUB1. Mean ± SD. (F) Sequence logo of amino acids P5-P5′ positions. (G) Cleavage of peptides containing SUB1 (red) and RAP1 (blue) sequences and alanine substitutions by rPMX. Mean ± SD.

Figure 3

WM382 and WM4 Target Engagement, Parasite Growth, Egress, and Invasion (A) Knockdown of PMIX and PMX expression in P. falciparum using glms ribozyme and glucosamine (GlcN). 3D7, 3D7-PMX_HA, and 3D7-PMIX_HA treated with GlcN and probed with anti-HA. (B) EC₅₀ for WM4 with P. falciparum in presence (light) and absence (dark) of 2.5 mM GlcN. Mean ± SD (eight experiments). (C) WM382 EC₅₀ for P. falciparum in presence (light) and absence (dark) of 2.5 mM GlcN. Mean ± SE (five experiments). (D) CETSA with WM4 and WM382. Immuno-blots probed with αHA in 3D7-PMIX_HA and 3D7-PMX_HA. Right: relative intensity at 55°C. (E) Time of parasite killing for WM5, WM4, and WM382 in blood stage. DMSO, control. Mean ± SD. (F) P. falciparum-infected erythrocytes with DMSO, WM4, or WM382. (G) WM4 and WM382 block merozoite egress (percentage). Mean ± SEM. (H) Stills of schizonts visualizing egress (20 nM WM4). Arrows, free merozoites. Supplemental Information includes Video S1 (control with no drug) and Video S2 (plus 20 nM WM4). (I) Parasites grown in WM4 or WM382 are shown and percentage parasitemia. Mean ± SD.

Figure 4

WM382 Cures Mice of P. berghei and P. falciparum Infection (A) WM382 suppresses P. berghei infection, orally administered twice daily (b.i.d. dosage) for 4 days (n = 4). WM382 (20 mpk) compared with 10 mpk chloroquine (CQ). ^∗∗p < 0.005: Mean ± SD. (B) P. berghei infected Swiss mice are cured with oral WM382. Mice (n = 4) treated for 4 days (b.i.d.) with WM382 (mpk) or chloroquine (10 mpk). (C) P. berghei infected mice (n = 4) cured with single, oral, daily doses (q.d.) of WM382. (D) Humanized NOD-scid IL2Rγ^null mouse model shows WM382 suppresses P. falciparum infection. Mice (n = 3), orally treated for 4 days (q.d.) with WM382 (mpk) or chloroquine (50 mpk). Mean ± SD. (E and F) Concentration of WM382 at 1, 2, 4, 6, and 24 h post first and last drug administration in blood from huSCID mice determined by LC-MS/MS, and (F) corresponding AUC values. Mean ± SD. Parasitemia monitored by fluorescence-activated cell sorting (FACS) and microscopy.

Figure 5

WM382 Prevents Transmission to Mosquitoes and Transition from Liver to Blood Infection (A) Oocyst counts from P. falciparum-infected mosquito guts for gametocytes treated with WM4 or WM382. Prevalence pies, proportions of mosquitoes with oocysts (black). Mean ± SD. (B) Mice were infected with five infectious mosquito bites (MB) or intravenous (i.v.) injection of 40,000 PbmCherryLuci sporozoites. From 52 h post infection (hpi), bioluminescence measured liver infection and egress, while bioluminescence and flow cytometry measured blood infection (Figure S6). (C) Bioluminescent images showing peak liver infection (52 hpi), liver egress (55 hpi), and blood infection (65 hpi) in WM382 treated and untreated mice. (D) Liver infection (52 hpi) was similar in all treatment groups and was reduced at 55 hpi. Mean ± SD. (E) Percentage loss of bioluminescence (egress) between 52 and 55 h. Mean ± SD. (F) Whole-body luminescence for blood infection at 65 hpi. Mean ± SD. (G) Blood parasitemia (FACS) at 65 hpi. Mean ± SD. (H) Time to patent blood infection for mice infected i.v. with sporozoites. (I) Time to patent blood infection following injection of 65 hpi supernatants from DMSO and WM382-treated P. berghei-infected HepG2 in vitro cultures. (J) Time-to-patent blood infection for mice infected with sporozoites by mosquito bite. Mean ± SD shown. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Data in H–J represent 5 to 15 mice per group. LOD, limit of detection.

Figure 6

PMIX and PMX Process Invasion Proteins (A) SERA5 processing inhibited by WM4 and WM382. (B) RAP1 processed by PMIX and SUB1 (left) and separated further for merozoites (right). (C) Merozoites probed with αASP inhibited by WM382. (D) PMIX autocatalytically processed and inhibited by WM382. (E) RON3 processing inhibited by WM382. (F) MTRAP processing inhibited by WM382. (G) SUB2 processing by PMX. (H) αAMA1 detection after treatment with E64, WM4, and WM382. Left: merozoites. Right: supernatant. (I) MSP1 detection (αMSP1/19) from merozoites treated with E64, WM4, and WM382. (J) PMX autocatalytically processed and inhibited by WM4 and WM382. (K) Rh1 processed by PMX. W2mef-HA parasites have three Rh1 genes with one HA tagged. (L). Rh2a/b are processed by PMX. Left: merozoites. Right: supernatants. Rh2a and Rh2b are identical but differ at C terminus (blue and gray domains). (M) Rh4 (αRh4) processed by PMX. A summary schematic for each protein is shown (HA-tag, yellow box; transmembrane, black box. Supernatants (Superⁿ or S) released from merozoites. M, merozoites. Position of detecting antibody signified by . Molecular weight kDa.

Figure 7

Rh5 Complex and EBA Proteins Processed by PMX or PMIX (A) Rh5 processed by PMX and inhibited by WM4 and WM382. Left: supernatant. Right: merozoites. (B) Ripr processed by PMX or PMIX inhibited by WM4 and WM382. Left: supernatant. Right: merozoites. (C) EBA140 processed by PMX and inhibited by WM4 and WM382. Left: αEBA140 monoclonal antibody (mAb) (2D6). Right: αEBA140 mAb (1H5). (D) EBA175 processed by PMX and inhibited by WM4 and WM382. (E) EBA175 processed by PMX and inhibited by WM4 and WM382. (F) EBA181 processed by PMX and inhibited by WM4 and WM382. (G) Summary of PMX function. A summary schematic for each protein is shown (HA-tag, yellow box; transmembrane, black box). Supernatants (Superⁿ or S) released from merozoites. M, merozoites. Position of detecting antibody signified by . C–F schematics show positions of F1 and F2 receptor binding domains. Molecular weight kDa.