Figure 2. Small molecules block AMA1-RON complex formation and inhibit merozoite invasion.

(a) Purified merozoites were used to test the effect of the three compounds on invasion of RBCs at 25 μM (white bars) and 50 μM (black bars) for 4 hr. Error bars show ± SEM from five experiments for NCGC00015280, NCGC00181034 and two for NCGC00014044. (b) Immunoprecipitation assay testing the ability of the inhibitors to block parasite AMA1-RON complex formation. Each inhibitor was used at 100 μM concentration and was immunoprecipitated using anti-RON4 antibody. RON2L peptide was used as a positive control. DMSO (1%), the solvent for the inhibitors, was used as a negative control. Experiments were performed twice and a representative western blot data is shown. (c) NCGC00015280 inhibits merozoite invasion of genetically distinct parasite clones. Purified schizonts from four different parasite clones were allowed to rupture and invade new RBCs for 4 to 6 hr in the presence of varying concentrations of the inhibitor. The number of newly invaded rings was measured by flow cytometry of SYBR green labeled parasites. IC₅₀: 12 μM (FVO), 14 μM (3D7), 13 μM (DD2) and 10 μM (HB3). Error bars show ± SEM from two experiments for each parasite clone. (d) Merozoite release from schizont-infected RBCs is not affected. The effect of the inhibitors on merozoite release was tested at 30 μM, the IC₅₀ for invasion. Error bars represent ± SEM from three experiments for NCGC00015280, NCGC00181034 and two for NCGC00014044. The number of parasites in the absence of inhibitor was considered 100%.