Fig. 6.

TH-PF01 suppressed parasite growth at different blood substages. (A) Glucose is essential for the survival of the blood-stage parasite. The P. falciparum (Dd2) grows only in the presence of glucose (peaking at 16 mM) or fructose (peaking at 50 mM). The physiological glucose concentration range in human blood was highlighted (3.9 to 6.9 mM). The RPMI media used for parasite culture contain 11 mM glucose (indicated by the vertical dotted line) and no fructose. The data represent two independent experiments and are shown as an average of three technical replicates. RFU: relative fluorescence units. Error bars represent SD. (B) The increased glucose consumption during the parasite developmental cycle was observed by monitoring glucose concentration in the culture medium (RPMI containing 11 mM glucose with 3% parasitemia and 1% hematocrit). The glucose concentration of two culture flasks containing tightly synchronized parasites (P. falciparum Dd2) and a flask containing uninfected RBC (uRBC) was monitored every 4 h, and parasite substages were assessed by microscopic observations. The data of parasite culture show the average of two flasks. The data of uRBC were calculated as the change of glucose concentration over 48 h. Error bars represent SD. (C) Basal ECAR shows that the schizont stage conducts the highest glycolysis among the substages. Early ring-, late ring-, trophozoite-, and schizont-stage parasites (PfDd2) in RBCs were seeded in an assay medium containing glucose (11 mM), and the basal ECAR was determined as the difference before and after 2-deoxy-d-glucose (2-DG, 50 mM as a final concentration) addition. Data were normalized with seeding density and parasitemia. All data were average values pooled from two to five independent experiments with three technical replicates. Error bars represent SEM. (D) TH-PF01 appeared as equipotent to all substages when the survival was assessed immediately after compound treatment; however, it required longer incubation time against ring-stage than late-stage parasites to show the same potency when incubated for an additional 36 h after washed. EC₅₀ values of TH-PF01 and dihydroartemisinin against substages were determined by lactate dehydrogenase (LDH) assay in the time course experiments depicted in E, i, ii, and iii. The horizontal dashed lines indicate the EC₅₀ value determined by a 72-h SYBR assay. The data show an average EC₅₀ with SD of two to five bioreplicates. (E) A schematic representation of the substage assay. Tightly synchronized parasites (Pf3D7) were exposed to TH-PF01 or dihydroartemisinin for various periods at the substages indicated. (F) Representative images of compound-treated parasites (SI Appendix, Fig. S5). Solid outline, TH-PF01 treated parasites; dotted outline, dihydroartemisinin-treated parasites.