Mechanism of antimalarial action of the synthetic trioxolane RBX11160 (OZ277)

Abstract

RBX11160 (OZ277) is a fully synthetic peroxidic antimalarial in clinical development. To study the possible mechanisms of action of RBX11160, we have examined its ability to inhibit PfATP6, a sarcoplasmic reticulum calcium ATPase and proposed target for semisynthetic peroxidic artemisinin derivatives. RBX11160 inhibits PfATP6 (apparent half-maximal inhibitory constant=7,700 nM) less potently than artemisinin (79 nM). Inhibition of PfATP6 is abrogated by desferrioxamine, an iron-chelating agent. Consistent with this finding, the killing of Plasmodium falciparum organisms by RBX11160 in vitro is antagonized by desferrioxamine. Artesunate and RBX11160 also act antagonistically against P. falciparum in vitro. A fluorescent derivative of RBX11160 localizes to the parasite cytosol in some parasites and to the food vacuole in other parasites. These data demonstrate that there are both similarities and differences between the antimalarial properties of RBX11160 and those of semisynthetic antimalarials such as artesunate and artemisinin.

FIG. 1.

RBX11160 inhibition of transporter activities. (a) Inhibition of PfATP6 activity by RBX11160 (50 μM) compared with results for control preparations (n = 6 for both groups; P = 0.0018). (b) Inhibition of mammalian SERCA by RBX11160 (100 μM; P = 0.13) and thapsigargin (10 μM; P = 0.0007) compared with results for control experiments. (c) Uptake of d-glucose in water-injected (diethyl pyrocarbonate [DEPC]) and PfHT-expressing oocytes is shown. There is no inhibition of PfHT activity by RBX11160 (50 μM, n ≥ 9 per column; P > 0.5). DMSO, dimethyl sulfoxide.

FIG. 2.

Apparent inhibitory constants for PfATP6 of RBX11160 and artemisinin. The apparent K_i values are 7,700 nM for RBX11160 (filled circles) (n = 3 for each value) and 79 nM for artemisinin (filled squares) (n = 5 for each value).

FIG. 3.

Effects of desferrioxamine on RBX11160. (a) Inhibition of PfATP6 activity by RBX11160 (gray column) (10 μM, n = 5) is abrogated by desferrioxamine (black column) (100 μM, P = 0.037, n = 9). (b) Isobologram of desferrioxamine and RBX11160. The summed fractional inhibitory concentration index (FIC) for this assay [geometric mean (range)] is 1.36 (0.97 to 2).

FIG. 4.

Immunofluorescence labeling of trophozoites. (a) Chemical structure of RBX11160-TAMRA. (b) Immunofluorescent staining of two parasites in a single erythrocyte by labeled RBX11160 (a). One trophozoite shows intense staining of the food vacuole (indicated by a long white arrow), whereas the other shows negative staining of the pigment-containing food vacuole (short white arrow) with diffuse cytosolic staining. (c) Bright-field view of panel b. Black arrows indicate parasite pigments in the food vacuole.

FIG. 5.

Confocal imaging of trophozoite stage. (a) Trophozoite labeled with ER-Tracker Blue showing cytosolic staining with negative staining of the food vacuole. (b) Corresponding bright-field image of panel a. (c) Parasite from panel a showing labeling with RBX11160-TAMRA. (d) Merged images (panels a to c).

FIG. 6.

Isobologram of RBX11160 and artesunate. Points lying above the line of additivity indicate antagonistic effects. The summed fractional inhibitory concentration index (FIC) for this assay [geometric mean (range)] is 2.4 (1.85 to 4.2).