Rosette-Disrupting Effect of an Anti-Plasmodial Compound for the Potential Treatment of Plasmodium falciparum Malaria Complications

Abstract

The spread of artemisinin-resistant parasites could lead to higher incidence of patients with malaria complications. However, there are no current treatments that directly dislodge sequestered parasites from the microvasculature. We show that four common antiplasmodial drugs do not disperse rosettes (erythrocyte clusters formed by malaria parasites) and therefore develop a cell-based high-throughput assay to identify potential rosette-disrupting compounds. A pilot screen of 2693 compounds identified Malaria Box compound MMV006764 as a potential candidate. Although it reduced rosetting by a modest 20%, MMV006764 was validated to be similarly effective against both blood group O and A rosettes of three laboratory parasite lines. Coupled with its antiplasmodial activity and drug-likeness, MMV006764 represents the first small-molecule compound that disrupts rosetting and could potentially be used in a resource-limited setting to treat patients deteriorating rapidly from malaria complications. Such dual-action drugs that simultaneously restore microcirculation and reduce parasite load could significantly reduce malaria morbidity and mortality.

Figure 1. Strain- and stage-dependent presentation of multiplets.

Cultures of (A) rosetting FCR3S1.2R and (B) non-rosetting NF54CSA parasites were co-stained with Hoechst and Dihydroethidium (DHE) to differentiate uninfected erythrocytes (uRBC) from ring- (R), trophozoite- (T) and schizont-infected (S) erythrocytes. The percentage of multiplets of each parasite stage were determined by gating for events that did not have a proportional forward scatter area (FSC-A) to forward scatter height (FSC-H) ratio. (C) The percentage of multiplets of each parasite is shown at different stages of parasite development (****P < 0.0001, N = 5). (D) Representative micrographs of multiplet-sorted (left three panels) and singlet-sorted (right-most panel) late-stage FCR3S1.2R parasites, with corresponding rosetting rate determined by microscopic enumeration of at least 300 parasites.

Figure 2. Correlation between rosetting rate and percentage of multiplets.

(A) Rosetting FCR3S1.2R parasites were treated for 2 h with various concentrations of heparin (0–5 mg/mL). Samples were split and analysed by microscopy to determine rosetting rate (100–200 iRBC) or by flow cytometry to determine percentage of multiplets (N = 4). (B) Rosetting FCR3S1.2R parasites were treated for 1 h with sera from 74 Cameroon patients and percentage of multiplets was determined by cytometry. Published values of the corresponding rosette-disruption effect in respective patient sera, determined by microscopy, were used to determine correlation.

Figure 3. Effect of antimalarial drugs on rosetting.

Rosetting trophozoites of (A) FCR3S1.2R, (B) PAvarO and (C) R29 were treated with various concentrations (between 0.01–10,000 μM) of atovaquone (AQ), artesunate (AS), chloroquine (CQ) or quinine (QN) for 12 h before being stained and analyzed by flow cytometry. Malaria culture media (MCM) and Heparin (10 mg/mL) controls from the start and at the end of the experiment were analyzed for comparison (*P < 0.05, ****P < 0.0001, N ≥ 3).

Figure 4. Effect of various rosette disruption methods on percentage of multiplets.

Rosetting FCR3S1.2R parasites cultured in O+ or A+ erythrocytes were subject to various rosette disruption methods and analysed by flow cytometry. Cells were (A) treated with heparin (N = 7), (B) co-incubated with anti-PfEMP1 IgG (N = 3) or (C) non-immune IgG (N = 3) or (D) mechanically disrupted by passage through 23G needle (N = 5).

Figure 5. Rosette-disruption properties of modified heparins and other glycosaminoglycans.

Rosetting FCR3S1.2R parasites grown in (A) O+ or in (B) A+ erythrocytes were treated for 2 h with 100 μg/ml of Bovine-sourced heparin (Hep Bov) that was unmodified (NS_2S_6S), 2-O-desulfated (NS_2__6S), partially 2-O, 6-O desulfated (NS_2$_6$), N-desulfated (N__2S_6S), 2-O, 6-O desulfated (NS_2__6_), N, 2-O desulfated (N__2__6S), N desulfated and partially 2-O, 6-O desulfated (N__2$_6$), N, 2-O, 6-O desulfated (N__2__6__), Heparan sulfate, Chondroitin sulfate A (CSA), Chondroitin sulfate C (CSC), Keratan sulfate, E. coli cell wall polysaccharide K5 (K5) or PBS control. Cells were analysed by flow cytometry and the percentage of multiplets after treatment are indicated (**P < 0.01, ***P < 0.001, ****P < 0.0001 when compared to DMSO control, N ≥ 3).

Figure 6. Hit validation and screening of ChemBridge compounds.

(A) Hits from the Prestwick, Asinex Protein-Protein Interaction (PPI) and Malaria Box library screens were re-screened at 10 μM together with selected compounds from the ChemBridge drug library (100 μM), DMSO (0.1%) and Heparin (10 mg/mL) controls. Rosetting FCR3S1.2R parasites cultured in A+ erythrocytes were stained and treated for 2 h with test 10 μM compounds before flow cytometry analyses. (B) Compound properties of MMV006764 from ChEMBL database are tabled here including molecular weight (MW), charge, Lipinsky rule of 5 violations (#Ro5), effective concentration 50% (EC₅₀) and inhibitory concentration 50% (IC₅₀) values.

Figure 7. Validation of MMV006764 rosette disruption activity.

Rosetting FCR3S1.2R parasites cultured in blood group A erythrocytes were treated for 2 h with various concentrations of (A) MMV006764 or (B) Heparin and the samples split for microscopic enumeration or (C) flow cytometry for correlation. (Between 150–300 late-stage parasites were counted in each microscopy sample, *P < 0.05, **P < 0.01, ****P < 0.0001, N = 3) (D) Cultures of rosetting FCR3S1.2R, PAvarO and R29 grown in blood group O+ or A+ erythrocytes were stained and treated with different concentrations of MMV006764 for 2 h before flow cytometry (**all points in box compared to untreated P < 0.01, N = 6, details of P-values in Supplementary Table S4). Rosettes of FCR3S1.2R grown in (E) blood group O+ or (F) A+ erythrocytes were dispersed by trituration and allowed to reform spontaneously in MCM, with or without 50 μM of MMV006764, MMV006656, AG-205/40776006 and ST017207, or 1 mg/ml of heparin.