Validation of the hexose transporter of Plasmodium falciparum as a novel drug target

Abstract

Chemotherapy of malaria parasites is limited by established drug resistance and lack of novel targets. Intraerythrocytic stages of Plasmodium falciparum are wholly dependent on host glucose for energy. Glucose uptake is mediated by a parasite-encoded facilitative hexose transporter (PfHT). We report that O-3 hexose derivatives inhibit uptake of glucose and fructose by PfHT when expressed in Xenopus oocytes. Selectivity of these derivatives for PfHT is confirmed by lack of inhibition of hexose transport by the major mammalian glucose and fructose transporters (Gluts) 1 and 5. A long chain O-3 hexose derivative is the most effective inhibitor of PfHT and also kills P. falciparum when it is cultured in medium containing either glucose or fructose as a carbon source. To extend our observations to the second most important human malarial pathogen, we have cloned and expressed the Plasmodium vivax orthologue of PfHT, and demonstrate inhibition of glucose uptake by the long chain O-3 hexose derivative. Furthermore, multiplication of Plasmodium berghei in a mouse model is significantly reduced by the O-3 derivative. Our robust expression system conclusively validates PfHT as a novel drug target and is an important step in the development of novel antimalarials directed against membrane transport proteins.

Fig. 1.

O-3 derivatives of d-glucose selectively inhibit PfHT. (a) The importance of the different d-glucose hydroxyl groups in interacting with Glut1 (hatched bars) and PfHT (black bars) transporters was assessed by testing different O-methyl-d-glucose analogues (10 mM) as competitors in uptake assays of d-glucose. (b) Uptake assays (mean ± SE of eight oocytes per condition) were carried out with d-[¹⁴C]glucose or d-[¹⁴C]fructose. Percentage of d-glucose or d-fructose uptake for each condition was compared with uptake in uncompeted oocytes (controls). All compounds were used at 10 mM except compound 3361 (1 mM). Hatched bars, Glut1; black bars, PfHT with d-glucose as substrate; white bars, PfHT with d-fructose as substrate. (c) Inhibition by compound 3361 of d-glucose uptake in PfHT (•) and Glut1 (▪) and d-fructose uptake in PfHT (○) and Glut5 (□).

Fig. 2.

Dependence on hexose concentrations of inhibition by compound 3361. (a Upper) Effect of compound 3361 on [³H]hypoxanthine incorporation in cultured parasites at different d-glucose (Left) and d-fructose (Right) concentrations. Red, blue, pink, orange, and green lines correspond respectively to 4, 6, 8, 10, and 12 mM glucose and 20, 25, 30, 35, and 40 mM fructose in culture medium. (Lower) Change in IC₅₀ for compound 3361 depending on the d-glucose (Left) and d-fructose (Right) concentrations. Data from three independent assays are shown (for glucose r² = 0.84 and P < 0.0001; for fructose r² = 0.80 and P = 0.0002). Values obtained with the multidrug-resistant strain K1 at extremes of d-glucose concentrations are represented by asterisks. (b) Effect of compound 3361 on the morphology of P. falciparum in culture (30μM compound 3361; 4 mM extracellular glucose) and of P. berghei in mice. Arrows show vacuole formation.