Plasmepsin IX/X (PMIX/X); Plasmodium

WM382 inhibits Plasmodium falciparum and P. vivax, with an IC50 value of 0.6 nM for P. falciparum, and exhibits considerable cytotoxicity against HepG2 cells (IC50=24.8 μM)[2][3]. With Ki values of 13.4 μM and 0.035 nM, respectively, WM382 binds PMV and PMX selectively[3]. The time to patent blood infection after injection is reminded by WM382 (1 nM and 100 nM) in P. berghei-infected HepG2 in vitro cultures[3].

In mouse models of P. berghei and P. falciparum parasites, WM382 (20 mg/kg twice daily or 1-30 mg/kg once day; po; for 4 d) is effective in eliminating infections. Asexual P. falciparum infection in humanized mice can be effectively treated with WM382, which also stops mosquito transmission[3].

Protease processing inhibition assays in P. falciparum parasites [2]
Processing inhibition assays were performed essentially as previously described (Favuzza et al., 2020). Protease inhibitors were added to synchronised late trophozoite/early schizont cultures, then passed over LD or LS magnetic columns to remove uninfected erythrocytes. WM4 and WM382 were added at 40 nM and 2.5 nM final concentrations respectively. A control dish without protease inhibitors was included. Parasites were eluted from columns with complete RPMI 1640 culture medium with inhibitor at the same concentration. Eluted parasites were adjusted to 5 × 106 schizonts/mL and 150 mL added per well of a 96-well flat-bottomed culture dish. The parasites were cultured for 16 h and a representative well smeared for Giemsa staining, to ensure that rupture had occurred normally (control well) or that rupture had been blocked (WM4, WM382 conditions). Parasites were centrifuged at 10000 g/10 min to collect merozoite and supernatant fractions. Proteins from both fractions were added to Reducing sample buffer and separated on 4-12% or 3-8% acrylamide gels for subsequent immunoblots.

---

Surface plasmon resonance [2]
PvPMX or PfPMX proteases were immobilised on a CM5 sensor chip by amine coupling in 10 mM actetate pH 5.5, injecting for 420 s at a flow rate of 10 mL min−1 typically immobilising around 4000 RU of protein. All experiments were performed in HBS-EP buffer (10 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% tween 20) at 18°C with a 30 mL min−1 flowrate. The compounds WM4 and WM382 were diluted to appropriate concentrations in the range 0.031 nM–16 nM in HBS-EP + buffer. Compounds were injected with a 90 s contact time and 1500 s dissociation, followed by 30 s regeneration with 50 mM glycine pH 9.5. Experiments were performed independently three times using a freshly immobilised sensor surface. PvPMX data were acquired on a BIAcore S200 instrument with data analysis in Biacore S200 evaluation software version 1.1 and PfPMX data collected on a BIAcore 8K+ instrument with data analysis in Biacore Insight evaluation software version 3.0.12.15655. All sensorgrams were double reference using an activated the ethanolamine blocked sensor surface and an HBS-EP + buffer blank. Affinities were determined using a 1:1 binding model fitting on and off rates for WM4, it was not possible to report affinities for WM382 as the off rate was too slow to determine.

---

CETSA Thermal Stability Assays [3]
Lysate CETSA experiments were conducted essentially as described (Dziekan et al., 2019). Samples for CETSA studies were prepared using highly synchronous schizont stage P. falciparum 3D7-PMIX_HA and 3D7-PMX_HA parasites. Late-stage parasites (40–44 h post-invasion) were purified by Percoll density gradient and incubated with 1 μM compound 1 (C1) inhibitor (synthesized in-house) (Hale et al., 2017). After 2–4 h incubation, mature schizonts were washed once with PBS and pelleted infected red blood cells were lysed in 20 volumes of 0.15% (w/v) saponin in PBS and incubated on ice for 20 min. Parasites were washed 3 times in ice-cold PBS and the final pellet was resuspended in 10 volumes of lysis buffer (0.4% NP-40 (Roche) / PBS) and lysed by three freezing (dry ice/ethanol bath) - thawing cycles. The lysates were cleared by centrifugation at 16,000 g for 30 min and the supernatant containing soluble parasite proteins kept at −80°C until use. Compounds 601 (5 μM), WM4 (2 μM) and WM382 (1 μM) were added to 8x 50 μg protein lysate aliquots (protein concentration measured by the BCA Protein Assay, incubated at room temperature (RT) for 3 min and heated at respective temperatures (temperature gradient 65-40°C) for 3min in a Biorad T100 thermocycler, followed by 3 min incubation at 4°C. The post-heating lysates were centrifuged at 13,000 g for 30 min at 4°C. Soluble proteins were resolved on precast 4%–12% gradient gels with MES running buffer according to the manufacturer’s directions.

Determination of EC50 in Knock down Parasite Lines [3]
Ring stage P. falciparum 3D7, 3D7-PMIX-HA and 3D7-PMIX-HA parasites were cultivated with increasing concentrations of GlcN (Sigma). After 72 h incubation at 37°C, trophozoite-infected erythrocytes were lysed in 0.06% saponin, pellets were solubilized in 2 times reducing SDS-PAGE sample buffer and analyzed by anti-HA immune-detection. EC50 of inhibition for WM4 and WM382 against P. falciparum 3D7, 3D7-PMIX-HA and 3D7-PMX-HA parasites, were determined in the absence and presence of 2.5 nM GlcN (normal and reduced protein expression of HA-tagged protein, respectively) by FACS determination and SYBR Green, as already described.

---

Time of Drug Killing[3]
3D7 parasite cultures were synchronized using 5% sorbitol (Sigma) twice at 46 h intervals then again when the culture was a mix of late schizonts and early rings. Triplicate 10ml cultures containing either 80nM WM4, 40nM WM5, 5nM WM382 or DMSO (Sigma) vehicle control were set up at 3% rings and 4% hematocrit. The parasitemia of each culture was quantitated every 8 h for 48 h by collecting 50 μL samples for counting by flow cytometry (as previously described). The developmental stage of the parasites was confirmed at each time point by microscopic examination of Giemsa stained thin blood films. Media and compound were replaced at the 32 h time point.

---

Invasion Assay[3]
3D7 parasite culture were synchronized by sorbitol treatment and WM4 (40 nM) and WM382 (2.5 nM), or DMSO control, added at ring stage. Late-stage parasites (> 40 h post invasion) were enriched by magnet separation and allowed to develop to fully segmented schizont-stage parasites. To prevent schizont rupture and merozoites release, control parasites were incubated with 1 μM compound 1 (C1). After 5–6 h of incubation, parasitophorous vacuole membrane enclosed merozoites (PEMS) were pelleted, resuspended in a small volume of complete culture medium (containing WM4, WM382 or C1) and filtered through a 1.2 μm syringe filters (Acrodisc; 32 mm; Pall). Filtrate containing purified merozoites was immediately added to fresh erythrocytes (70%–80% hematocrit), incubated in a shaker (1,100 rpm) at 37°C for 20 min to allow invasion of host cells, and then diluted to 2% hematocrit. After 24 h incubation at 37°C, invasion was evaluated by measuring parasitemia by microscopy (Giemsa-stained thin smears) and flow cytometry

---

Determination of Time to Resistance[3]
For experiment 1, cultures of 105, 106, 107, 108 and 109 Dd2 parasites were exposed to either 10 nM atovaquone or 1.5 nM WM382 and monitored for 90 days. For experiment 2, triplicate cultures of 106, 107 and 108 Dd2 parasites were exposed to either 5 nM atovaquone, 80 nM WM5 or 1.5 nM WM382 and monitored for 62 days. Each experiment was monitored by weekly microscopic examination of thin blood films. Media and compound were replaced three times each week.

Animal/Disease Models: Mice infected with P. berghei[3]
Doses: 20 mg/kg
Route of Administration: po (oral gavage); twice (two times) daily for 4 days; monitored for 30 days
Experimental Results: Cured mice of P. berghei and prevents blood infection from the liver.

---

Animal/Disease Models: Humanized nonobese diabetic-severe combined immunodeficiency (NOD-scid) IL2Rgnull mouse model (NSG)[3]
Doses: 1, 3, 10, 30 mg/kg
Route of Administration: po (oral gavage); one time/day for 4 days; monitored for 7 days
Experimental Results: Cleared of parasitemia by day 6 at 30 mg/kg or day 7 at 3 and 10 ma/kg.

---

Dose Ranging Test for P. berghei Infection in Mice [3]
In the dose ranging studies, mice were treated orally with WM382 for 4 days by a b.i.d. dosing regimen at 30, 10, 3 or 1 mpk/day (Figure 4B), or by a q.d. dosing regimen at 60, 30 or 10 mpk/day (Figure 4C), with the first dose given 2 h after infection. Control mice were treated orally with chloroquine for 4 days under q.d. dosing regimen at 10 mpk/day. From day 2 to 30 post infection, parasitemia was measured daily by flow cytometry and microscopy, as described above. Survival of animals to day 30 post infection, with no detectable parasites in the peripheral blood, were considered to be cured (i.e., 100% efficacy).

---

P. falciparum Humanized NOD-scid IL2R_null Mouse Model [3]
Compounds were tested in the murine P. falciparum SCID model (Angulo-Barturen et al., 2008). Briefly, WM382 formulated in 20% DMSO, 60% propylene glycol and 20% water, was administered to a cohort of age-matched female immunodeficient NOD-scid IL- 2Rγnull mice previously engrafted with human erythrocytes. The mice were intravenously infected with 2 × 107 P. falciparum Pf3D7-infected erythrocytes (day 0) (Angulo-Barturen et al., 2008). On day 3 after infection, mice were randomly allocated to treatments that were administered once a day for 4 consecutive days (n = 3 mice) by oral gavage at 10 mL/kg. Parasitemia was measured by microscopy. Chimerism was monitored by flow cytometry using anti-murine erythrocyte TER119 monoclonal antibody and SYTO-16 and then analyzed by flow cytometry in serial 2 μL blood samples taken every 24 h until assay completion.

---

In Vivo Analysis of P. berghei Liver Parasite Growth and Transition to Blood Infection [3]
Female BALB/c mice were infected with PbmCherryLuci sporozoites by either intravenous injection or infectious mosquito bite challenge. Mice (infected by either route) received either no treatment or were treated orally with WM382 (prepared as previously described) at doses and h post infection (hpi) as indicated. For i.v. injections, freshly isolated salivary gland sporozoites were filtered through glass wool to remove debris before 40,000 sporozoites were resuspended in 200 μL Schneider’s Insect Media immediately prior to injection. For infection by mosquito bite, the percent of mosquitoes that contained oocysts was used to place 5 infected mosquitoes in individual feeding cups (for example, if 83% of mosquitoes in the batch had oocysts then 6 mosquitoes were placed in each cup). Mice were anesthetised using ketamine/xylazine and placed on individual feeding cups to begin the infection. Mosquitoes were allowed to feed on the mice for 15 min and mice were rotated between feeding cups every min to promote probing and ensure that all mice were equally exposed to infectious mosquito bites. From day 3 – 30 post infection mice were monitored daily for the presence of parasites in Giemsa stained thin blood smears and considered to be protected from blood infection if they remained negative through this period.

---

Peters’ 4-Day Suppressive Test for P. berghei Infection in Mice [3]
For results shown in Figure 1 A male Swiss mice were infected intraperitoneally (IP) with 1 × 106 P. berghei ANKA-parasitised red blood cells withdrawn from a previously infected donor mouse. Test compounds were prepared in a vehicle consisting of 10% DMSO/90% Solutol (5% Solutol® HS-15 in 0.9% saline). Two h post infection, mice were treated on 4 consecutive days (q.d. regimen, once a day) with an IP dose of test compounds (WM4, WM5: 20 mpk) or chloroquine (10 mpk), or received an IP injection of vehicle as a control. Peripheral blood samples were taken 24 h after administration of the last dose, and parasitemia was measured by microscopic analysis of Giemsa-stained blood smears. Parasitemia values were averages for 6 mice per group and are expressed as percent parasitemia. For results shown in Figure 4A ‘donor’ female Swiss mice were infected intraperitoneally (IP) with blood stage P. berghei parasites constitutively expressing GFP (P. berghei ANKA GFPcon 259cl2 (Franke-Fayard et al., 2004). Three days later, groups of 4 ‘acceptor’ Swiss mice were infected intravenously (IV) with 1 × 107 parasitised erythrocytes from the ‘donor’ mice. Two h post infection, experimental mice (4 per cohort) were left untreated (control mice) or treated orally on 4 consecutive days with test drugs formulated in 20% DMSO/60% PG/20% water (v/v/v) or chloroquine dissolved in water. Mice were treated with WM382 for 4 days by a b.i.d. dosing regimen (twice a day) at 20 mpk/day, with the first dose given 2 h after infection. Peripheral blood samples were taken 12 h after the last treatment, and parasitemia measured by flow cytometry (proportion of GFP-positive cells in 100,000 recorded events using FACSCalibur, BD) and microscopic analysis of Giemsa-stained blood smears. Parasitemia values were averages for 4 mice per group and are expressed as percent parasitemia.

Oral bioavailability (F=8% in rat and 38% in mice)

Plasma protein binding = 95%

[1]. Manuel de LR, et al. The Invention of WM382, a Highly Potent PMIX/X Dual Inhibitor toward the Treatment of Malaria. ACS Med Chem Lett. 2022 Oct 12.
[2]. Hodder AN, et al. Basis for drug selectivity of plasmepsin IX and X inhibition in Plasmodium falciparum and vivax. Structure. 2022 Jul 7;30(7):947-961.e6.
[3]. Favuzza P, et al. Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle. Cell Host Microbe. 2020 Apr 8;27(4):642-658.e12.

Drug resistance to first-line antimalarials─including artemisinin─is increasing, resulting in a critical need for the discovery of new agents with novel mechanisms of action. In collaboration with the Walter and Eliza Hall Institute and with funding from the Wellcome Trust, a phenotypic screen of Merck’s aspartyl protease inhibitor library identified a series of plasmepsin X (PMX) hits that were more potent than chloroquine. Inspired by a PMX homology model, efforts to optimize the potency resulted in the discovery of leads that, in addition to potently inhibiting PMX, also inhibit another essential aspartic protease, plasmepsin IX (PMIX). Further potency and pharmacokinetic profile optimization efforts culminated in the discovery of WM382, a very potent dual PMIX/X inhibitor with robust in vivo efficacy at multiple stages of the malaria parasite life cycle and an excellent resistance profile.[1]

---

Plasmepsins IX (PMIX) and X (PMX) are essential aspartyl proteases for Plasmodium spp. egress, invasion, and development. WM4 and WM382 inhibit PMIX and PMX in Plasmodium falciparum and P. vivax. WM4 inhibits PMX, while WM382 is a dual inhibitor of PMIX and PMX. To understand their function, we identified protein substrates. Enzyme kinetic and structural analyses identified interactions responsible for drug specificity. PMIX and PMX have similar substrate specificity; however, there are distinct differences for peptide and protein substrates. Differences in WM4 and WM382 binding for PMIX and PMX map to variations in the S' region and engagement of the active site S3 pocket. Structures of PMX reveal interactions and mechanistic detail of drug binding important for development of clinical candidates against these targets.[2]

---

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.[3]

C29H36N4O4

504.620547294617

504.273

C, 69.02; H, 7.19; N, 11.10; O, 12.68

2606990-92-3

154699453

Typically exists as solid at room temperature

3.4

2

5

5

37

900

2

CCC1(CC(=O)N(C(=N1)N)[C@@H]2CCOC3=C2C=C(C=C3)C(=O)N[C@H]4CC(OC5=CC=CC=C45)(C)C)CC

ZSZSSSHEMYULPX-FCHUYYIVSA-N

InChI=1S/C29H36N4O4/c1-5-29(6-2)17-25(34)33(27(30)32-29)22-13-14-36-23-12-11-18(15-20(22)23)26(35)31-21-16-28(3,4)37-24-10-8-7-9-19(21)24/h7-12,15,21-22H,5-6,13-14,16-17H2,1-4H3,(H2,30,32)(H,31,35)/t21-,22+/m0/s1

(4R)-4-(2-amino-4,4-diethyl-6-oxo-5H-pyrimidin-1-yl)-N-[(4S)-2,2-dimethyl-3,4-dihydrochromen-4-yl]-3,4-dihydro-2H-chromene-6-carboxamide

WM-382; WM382; 2606990-92-3; CHEMBL5171401; (4R)-4-(2-amino-4,4-diethyl-6-oxo-5H-pyrimidin-1-yl)-N-[(4S)-2,2-dimethyl-3,4-dihydrochromen-4-yl]-3,4-dihydro-2H-chromene-6-carboxamide; (4R)-4-[(2E)-4,4-diethyl-2-imino-6-oxo-1,3-diazinan-1-yl]-N-[(4S)-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3,4-dihydro-2H-1-benzopyran-6-carboxamide; I0L; SCHEMBL22997111; GTPL11162; WM 382

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 5 mg/mL (9.91 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)