Fig 1.
IC50 determination of glucose uptake by PfHT-overexpressing HEK293 cells.
Uptake of 2-DG by HEK293 cells overexpressing PfHT at increasing concentrations of the indicated compounds. IC50s were calculated using nonlinear regression analysis IC50 determinations using GraphPad Prism 6.0. Uptake data are expressed as means ± SEMs (n = 3). Chemical structures of the compounds tested are shown for comparison.
Fig 2.
Specificity of hit compounds for PfHT over class I human orthologues.
Uptake of 2-DG by HEK293 cells overexpressing hGLUT1-4 in the presence of the indicated compounds at indicated concentrations approximating their IC50 for PfHT as shown in Fig 1. CB was used as a positive control.
Fig 3.
IC50 determination of glucose uptake by HEK293 cells overexpressing human class I orthologues.
2-DG uptake in response to increasing WU-1 (A) or WU-2 (B) concentrations in HEK293 cells expressing the indicated GLUT protein. IC50s were calculated using nonlinear regression analysis using GraphPad Prism 6.0 and are tabulated in Table 1. Uptake data are expressed as means ± SEMs of three independent experiments performed in triplicate.
Table 1.
IC50 concentrations and maximal inhibition for WU-1 and WU-2 for class I GLUT overexpressing cells.
Fig 4.
Inhibition of growth and IC50 determination of glucose uptake by isolated P. falciparum parasites.
A) IC50s for inhibition of growth of P. falciparum strain 3D7 intraerythrocytic forms by each compound were determined via a growth inhibition assay as described in Materials and Methods. Data are expressed as means ± SEMs of three independent experiments performed in duplicate. B) Uptake of 2-DG by isolated P. falciparum trophozoites at increasing concentrations of WU-1. Distribution ratios (i.e., the ratio of intracellular concentration of radiolabel relative to the extracellular concentration) were calculated as described previously [23, 50]. IC50s were calculated using nonlinear regression analysis (GraphPad Prism 6.0). Uptake data are expressed as means ± SEMs of three independent experiments performed in triplicate.
Fig 5.
IC50 determination of hexose uptake by HEK293 cells overexpressing representative human class II and III orthologues.
A) Uptake of 2-DG by HEK293 cells overexpressing either PfHT or class III GLUT transporter, hGLUT8. B) Uptake of [3H]-D-fructose (Frc) in HEK293 cells overexpressing hGLUT8, or PfHT or class II transporter, GLUT5. Uptake data are expressed as means ± SEMs of three independent experiments performed in triplicate. IC50s were calculated using nonlinear regression analysis (GraphPad Prism 6.0) and are tabulated in Table 2.
Table 2.
IC50 concentrations and selectivity indices for WU-1 in GLUT5 and GLUT8 overexpressing cells.
Fig 6.
C-1 is a non-competitive inhibitor of PfHT.
HEK293PfHT cells were incubated with increasing concentrations of [3H]-D-fructose (Frc). Data were fit by non-linear regression analysis using GraphPad Prism 6.0 software. Uptake data are expressed as means ± SEMs (n = 3). Dixon plot shows WU-1 is a noncompetitive inhibitor with a Ki of 4.4 ± 0.3 μM.
Fig 7.
PfHT mediated uptake of [3H]-D-glucose and [3H]-L-glucose into liposomes.
Purified PfHT protein (± FLAG tag) was reconstituted into eggPC/POPA/POPE (70:15:15) liposomes. A) SDS-PAGE of PfHT liposomes stained with Blue Bandit protein stain. B) Immunoblot analyses of PfHT liposomes using an anti-FLAG antibody (Ab) and a C-terminal PfHT specific antibody. (±) TEV indicates whether the FLAG tag of purified FTPfHT protein was cleaved off with TEV protease prior to the reconstitution into liposomes. C. Saturable, zero-trans uptake of [3H]-D-glucose into PfHT (-FLAG) liposomes (blue) compared to nonspecific uptake of [3H]-L-glucose (black). Uptake data were fitted using nonlinear regression analysis (GraphPad 6.0). Data are expressed as means ± SEMs (n = 3).
Fig 8.
WU-1 directly inhibits both the binding of the glucose analogue (ATB-BMPA) to PfHT and PfHT transport activity.
A) Biotinylated ATB-BMPA, a bis-mannose containing photolabel, binds to the glucose binding site of PfHT. Liposomes containing 0.65 μg PfHT were preincubated with ± 10 μM WU-1 prior to labeling with biotinylated ATB-BMPA. PfHT bound with biotinylated ATB-BMPA was analyzed by immunoblot analysis using a fluorescently labeled streptavidin (Strep). The strep signal was normalized to the amount of PfHT protein. Data are expressed as the means ± SEM., n = 3; (*), p<0.05 vs. vehicle control as determined by the paired Student’s t test. B) WU-1 inhibits the specific uptake ([3H]-D-glucose minus ([3H]-L-glucose) into PfHT-containing liposomes. Different concentrations of WU-1 were added to the liposomes 20 min prior to the initiation of the transport reaction. Uptake (quenched after 50 sec) was normalized to the amount of PfHT in the liposomes. Data were fit by nonlinear regression analysis using GraphPad Prism 6.0 software to calculate the IC50 for WU-1. Data are expressed as mean ± SEM of three independent experiments.
Fig 9.
Effect of the FLAG tag on the inhibition of PfHT-mediated glucose uptake by WU-1, CB, and ethylideneglucose.
A) Inhibition of 2-DG uptake by WU-1 in HEK293 cells overexpressing PfHT and FTPfHT. B) Inhibition of 2-DG uptake by the endofacial ligand cytochalasin B (CB) in HEK293 cells overexpressing PfHT and FT-PfHT. C) Inhibition of 2-DG uptake by the exofacial ligand ethylideneglucose in HEK293 cells overexpressing PfHT and FTPfHT. Data (Panels A-C) were fit by nonlinear regression analysis using GraphPad Prism 6.0 software to calculate the IC50s. Data are expressed as means ± SEMs of three independent experiments performed in triplicate. D) Kinetic analysis showed that the FLAG-tag had little effect on the Km. 2-DG uptake (1 min) were measured in HEK293 cells overexpressing PfHT and FTPfHT at different 2-DG concentrations. Km values were determined from nonlinear regression analysis using GraphPad Prism 6.0. Data are plotted in double-reciprocal form and expressed as the means ± SEMs of three independent experiments performed in triplicate.
Fig 10.
Simple carrier model illustrating the transport of glucose by an alternating access mechanism.
Glucose can access the glucose binding site of PfHT from either the exofacial side or the endofacial side of the membrane but not simultaneously. N and C represent the N-terminal and C-terminal α-helical bundles of PfHT. Upon binding glucose, PfHT undergoes a series of conformational changes that allows the translocation of glucose through the central pore and its release from PfHT at the other side of the membrane. Transport is bidirectional and down the concentration gradient.