Fig. 5.

TH-PF01 derivatives selectively target PfHT1. (A) PfHT1 inhibitors showed a reasonable correlation between blood-stage parasite growth inhibition (EC₅₀) and biochemical inhibition (IC₅₀) of PfHT1 glucose transport activity. (B) Glucose concentration in culture media offsets EC₅₀ values of TH-PF01 and TH-PF03 in a dose–response manner but not common antimalarial drugs quinine, mefloquine, and dihydroartemisinin. The blood-stage P. falciparum Dd2 was exposed to the test compounds in assay media containing glucose at different concentrations for 48 h starting at the ring stage, and the parasite growth was determined by SYBR Green I. The data represent four (TH-PF01) or two (TH-PF03, quinine, mefloquine, and dihydroartemisinin) independent experiments and are shown as average ± SD of two technical replicates. CM: culture medium. (C) The inhibition of glycolytic activity by TH-PF01 was observed by Seahorse extracellular flux analyzer. Dd2 schizont-stage parasites in RBCs were seeded in medium without glucose and exposed to glucose (11 mM as final concentration) or fructose (40 mM as final concentration) at 15 min (the first vertical dotted line), resulting in robust increases of ECAR. The addition of TH-PF01 (20 µM as final concentration) or 2-deoxy-d-glucose (2-DG) (50 mM as the final concentration) at 61 min (the second vertical line) lowered the ECAR, indicating glycolytic activity was inhibited. Data were normalized with ECAR values before and after the glucose additions as 0 and 100%, respectively. All data were average values pooled from two independent experiments with three technical replicates. Error bars represent SEM. (D) Extracellular flux analysis showed that TH-PF01 and TH-PF03 inhibit glycolytic activity in a dose-dependent manner in the early (rings) and late stages (trophozoites/schizonts) of the parasites in RBCs as well as freed late stages from RBCs. The Dd2 parasites were seeded in the assay medium containing glucose (11 mM), and TH-PF01 or TH-PF03 was added four times in sequence at the final concentrations of 0.4, 1, 2.5, and 6.25 or 0.2, 0.5, 1.25, and 3.13 µM. Glycolysis inhibitor, 2-DG, was added at 50 mM once. ECAR values were normalized with the values before the first compound addition as 100% and the values of background as 0%. All data were average values pooled from two independent experiments with two or three technical replicates. Error bars represent SEM. (E) The glucose concentration in the assay media and the potency of TH-PF01 against the glycolysis activity show a negative correlation. PfDd2 schizont-stage parasites in RBCs were seeded in an assay medium supplemented with glucose at 11, 5.5, or 2.75 mM, and TH-PF01 was sequentially added four times with the final concentrations of 0.4, 1, 2.5, and 6.25 µM. ECAR values after compound addition were normalized with the value before the first compound addition as 100% and the value of background as 0%. All data were average values pooled from two independent experiments with two technical replicates. Error bars represent SEM.