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. 2016 Jul 15;11(7):1908-16.
doi: 10.1021/acschembio.6b00304. Epub 2016 May 11.

Inhibitor Discovery for the Human GLUT1 from Homology Modeling and Virtual Screening

Peter Man-Un Ung  1 , Wenxin Song  2 , Lili Cheng  2 , Xinbin Zhao  2 , Hailin Hu  2 , Ligong Chen  2 , Avner Schlessinger  1   3
Affiliations

Inhibitor Discovery for the Human GLUT1 from Homology Modeling and Virtual Screening

Peter Man-Un Ung et al. ACS Chem Biol. .

Abstract

The human Glucose Transporter 1 (hGLUT1 or SLC2A1) is a facilitative membrane transporter found in the liver, intestines, kidney, and brain, where it transports sugars such as d-glucose and d-galactose. Genetic variations in hGLUT1 are associated with a broad range of diseases and metabolic disorders. For example, hGLUT1 is upregulated in various cancer types (e.g., breast carcinoma) to support the increased anaerobic glycolysis and the Warburg effect. Thus, hGLUT1 is an emerging therapeutic target, which also transports commonly used cancer biomarkers (e.g., (18)F-DG). In this study, we use computational prediction followed by experimental testing, to characterize hGLUT1. We construct homology models of hGLUT1 in a partially occluded outward open ("occluded") conformation based on the X-ray structure of the E. coli xylose transporter, XylE. Comparison of the binding site of the occluded models to experimentally determined hGLUT structures revealed a hydrophobic pocket adjacent to the sugar-binding site, which was tested experimentally via site-directed mutagenesis. Virtual screening of various libraries of purchasable compounds against the occluded models, followed by experimental testing with cellular assays revealed seven previously unknown hGLUT1 ligands with IC50 values ranging from 0.45 μM to 59 μM. These ligands represent three unique chemotypes that are chemically different from any other known hGLUT1 ligands. The newly characterized hydrophobic pocket can potentially be utilized by the new ligands for increased affinity. Furthermore, the previously unknown hGLUT1 ligands can serve as chemical tools to further characterize hGLUT1 function or lead molecules for future drug development.

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Figures

Figure 1
Figure 1. Crystal structures of hGLUT1 and E. coli XylE
Shown is side view of (A) hGLUT1 in an inward-open conformation (4PYP) and (B) XylE in a partially occluded outward open conformation (4GBZ). The occluded XylE structure has more compact ligand binding site and protein-ligand interactions than the inward-open hGLUT1 structure.
Figure 2
Figure 2. The hydrophobic H-pocket adjacent to the hGLUT1 sugar-binding site
Homology model of hGLUT1 based on the occluded XylE structure is in white and inward-open hGLUT1 structure is shown in gold color. (A) The hydrophobic moiety of multiple docked ligands (e.g., PUG-1) is predicted to reside in the H-pocket lined by the hydrophobic residues G27, T30, I164, V165, I168, and F291 (yellow sticks). (B) The H-pocket is larger in the hGLUT1 model in the occluded conformation (green spheres; 37 Å3) than in (C) the inward-open hGLUT1 structure (magenta spheres; 10 Å3) due to the rearrangement of the transmembrane helices (TM1, TM5, and TM7). (D) The occluded H-pocket (green surface) is larger and shaped differently than the inward-open H-pocket (purple spheres).
Figure 3
Figure 3
Western blots of (A) hGLUT1 in whole cell lysate and (B) biotinylated hGLUT1 in membrane protein fractions prepared from CHO cells stably transfected with empty vector (EV), wild-type (WT), and mutated hGLUT1 plasmids. Na+/K+-ATPase and β-actin were used as plasma membrane and cytoplasmic protein markers, respectively. (C) Specific [3H]-2-deoxy-D-glucose uptake of WT and mutant hGLUT1 in stably transfected CHO cell lines. Experiments were performed in triplicate. Error bars represent SEM.
Figure 4
Figure 4. Newly discovered ligands of hGLUT1
Predicted binding mode of (A) PUG-1 and (B) PUG-4 with the hGLUT1 models in the occluded conformation. The ligands (green sticks) occupy similar space to that of the sugar in the XylE template structure, and make critical hydrogen bonds with key residues (yellow dotted lines). An alternative rotamer of F291 was also used in the virtual screening (gold sticks). These ligands inhibit radiolabeled 2-DG uptake with significant IC50 values, as measured in a CHO-hGLUT1 cell line.
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