[3H]-2-deoxy-D-glucose ([3H]-2-DG) Uptake Assay

The stably transfected CHO cells were seeded in poly 24-well plates and were grown to 80–90% confluence. Cells were rinsed with PBS buffer and incubated in 280 μL of PBS buffer containing 61.73 μM [³H]-2-DG (PerkinElmer, Boston, MA) in the presence and absence of 50 μM test compound for 5 mins. The known hGLUT1 inhibitor phloretin (Accela ChemBio, San Diego, CA) was included as a positive control. Unradiolabelled 2-DG (40 mM) (Sigma, St. Louis, MO) was included in order to calculate specific inhibition of predicted inhibitors. Compounds were obtained from the National Cancer Institute (NCI) or purchased from commercial vendors. The reaction was terminated by washing cells twice with ice-cold PBS, followed by addition of 200 μL lysis buffer (0.1% (w/v) SDS, 0.1 N NaOH). Intracellular radioactivity was determined by scintillation counting. CHO-hGLUT1 cells showed a 3.92-fold increase in probe uptake relative to the CHO-EV cells and demonstrated to be a suitable system for sugar uptake inhibition assay (Fig. S2B). Probe-independent specific inhibition of the tested compound, which excludes hGLUT1 inhibition due to [³H]-2-DG, was calculated as follow:

where R is the intracelullar radioactivity of the treatment group, R_2DG is the intracellular radioactivity of [³H]-2-DG treated group, and D₀ is the intracellular radioactivity of the control group of [³H]-2-DG. Dose-dependent inhibition was measured under the same conditions as the single-point measurements. Cells were incubated with compounds at 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, and 3000 μM. Ligand efficiency (LE) of the compounds was calculated with the following equation:

where R is the ideal gas constant, T is temperature at 25 °C, and HA is the number of heavy atoms.