Fig 1.
Flow chart for screen of TCAMS library.
The TCAMS library of 13,533 compounds with demonstrated growth inhibitory activity against intraerythrocytic Plasmodium falciparum parasites was screened by sequential criteria. The steps included: 1) proliferation inhibitory screen (>65% inhibition at 3 μM concentration or >20% differential inhibition among the three strains) of PfHT, LmxGT2, and GLUT1 reporter strains to produce 401 primary hits; 2) 96-well plate assays for compounds (20–30 μM) that inhibited uptake of 200 μM [3H] D-glucose by ≥90%; 3) individual uptake assays for compounds (10 μM) that inhibited glucose uptake by ≥50%; 4) individual uptake assays for compounds (10 μM) that inhibited uptake of 100 μM [3H] L-proline by ≤10%; 5) dose-response curves for compounds that selectively inhibited uptake of glucose through PfHT versus GLUT1 (1 compound plus 1 additional hit that emerged from analysis of analogs). Numbers in parentheses represent the number of positive hits obtained after each sequential step.
Fig 2.
Compounds from the TCAMS (1–6) and Malaria Box (7–9) libraries that were selective inhibitors of glucose, but not proline, transport.
Dose-response curves for inhibition of uptake of 100 μM [3H] D-glucose by each compound were performed for PfHT and GLUT1, and the IC50 values are tabulated. SIglucose indicates Specificity Index for glucose uptake and is IC50 for GLUT1/IC50 for PfHT. The reported EC50 values (PubChem web site, http://pubchem.ncbi.nlm.nih.gov/) for inhibition of growth of P. falciparum strain 3D7 intraerythrocytic forms by each compound are also tabulated.
Fig 3.
Dose-response curves for inhibition of glucose uptake by PfHT and GLUT1.
Compounds 1 (A), 7 (B), and 13 (C) were applied over a range from 10-9–10-5 M to Δlmxgt1-3 null mutants expressing either PfHT (filled circles) or GLUT1 (open circles), and uptake of 100 μM [3H] D-glucose was measured in a 1 min uptake assay. Results are plotted as the mean and standard deviation (error bars) from 3 replicate uptake determinations. Data were fitted to a sigmoidal inhibition curve.
Fig 4.
Analogs of Compound 1 were tested for inhibition of uptake of 100 μM [3H] D-glucose by PfHT and GLUT1, as in Fig 2.
NI indicates no inhibition, and NA indicates does not apply. Other abbreviations are as in Fig 2.
Fig 5.
Inhibition of uptake of 100 μM [3H] D-glucose (Glc, black bars), 12.5 μM [3H] hypoxanthine (Hyp, stippled bars), 1 μM [3H] uridine (Urd, white bars), and 100 μM [3H] L-proline (Pro, square-hatched bars) by Compounds 1 (A), 7 (B), and 13 (C).
Concentrations of each inhibitor are shown on the x-axis; DMSO represents the control using the DMSO vehicle without any inhibitor. Proline uptake was not measured at 0.1 or 1.0 μM concentrations of inhibitors, hence the cross-hatched bar representing proline uptake does not appear for these concentrations.
Fig 6.
Analogs of the low potency, low specificity inhibitor Compound 8 were tested for inhibition of uptake of 100 μM [3H] D-glucose by PfHT and GLUT1, as in Fig 2.
Fig 7.
Inhibition of glucose uptake by PfHT by Compounds 1, 7, and 13.
Substrate saturation curves (A, B, C) were performed for PfHT in the presence of various concentrations of Compounds 1, 7, and 13, respectively. Data represent the mean and standard deviation of 3 replicate uptake assays. Data were fitted to the Michaelis-Menten equation by non-linear regression.
Table 1.
The kinetics of inhibition of glucose uptake by Compounds 1, 7, and 13.
Table 2.
Inhibition of proliferation of malaria parasites and of human foreskin fibroblasts by Compounds 1, 10, 12, and 13 and control compounds.
Table 3.
In vitro absorption-distribution-metabolism-excretion (ADME) properties of Compounds 1, 10, 12, 13 and control compounds.