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. 2016 Apr 26;113(17):4711-6.
doi: 10.1073/pnas.1603735113. Epub 2016 Apr 12.

Mechanism of Inhibition of Human Glucose Transporter GLUT1 Is Conserved Between Cytochalasin B and Phenylalanine Amides

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Free PMC article

Mechanism of Inhibition of Human Glucose Transporter GLUT1 Is Conserved Between Cytochalasin B and Phenylalanine Amides

Khyati Kapoor et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Cancerous cells have an acutely increased demand for energy, leading to increased levels of human glucose transporter 1 (hGLUT1). This up-regulation suggests hGLUT1 as a target for therapeutic inhibitors addressing a multitude of cancer types. Here, we present three inhibitor-bound, inward-open structures of WT-hGLUT1 crystallized with three different inhibitors: cytochalasin B, a nine-membered bicyclic ring fused to a 14-membered macrocycle, which has been described extensively in the literature of hGLUTs, and two previously undescribed Phe amide-derived inhibitors. Despite very different chemical backbones, all three compounds bind in the central cavity of the inward-open state of hGLUT1, and all binding sites overlap the glucose-binding site. The inhibitory action of the compounds was determined for hGLUT family members, hGLUT1-4, using cell-based assays, and compared with homology models for these hGLUT members. This comparison uncovered a probable basis for the observed differences in inhibition between family members. We pinpoint regions of the hGLUT proteins that can be targeted to achieve isoform selectivity, and show that these same regions are used for inhibitors with very distinct structural backbones. The inhibitor cocomplex structures of hGLUT1 provide an important structural insight for the design of more selective inhibitors for hGLUTs and hGLUT1 in particular.

Keywords: GLUT inhibitor; X-ray structure; cytochalasin B; glucose facilitator; human MFS transporter.

Conflict of interest statement

Conflict of interest statement: R.C.H, P.B., I.H., T.M., and H.S. are or have been employees and stockholders of Bayer AG.

Figures

Fig. 1.
Fig. 1.
Chemical structures of inhibitors used in this study and their binding sites in hGLUT1. Cytochalasin B (A), GLUT-i1 (B), and GLUT-i2 (C) were used for cocrystallization with WT-hGLUT1. (D) Cartoon of hGLUT1 viewed cytoplasmic side down, with bound cytochalasin B shown as red sticks in the central cavity. Helices are shown as rods labeled using the conventional numbering scheme for major facilitator superfamily transporters, and are color-coded as follows: N-terminal domain, yellow; C-terminal domain, orange; intracellular (IC) domain, green.
Fig. S1.
Fig. S1.
Glycosidase digestion pattern of hGLUT1 expressed in yeast. Lane 1, protein marker; lane 2, undigested; lane 3, endoglycosidase H (EndoH); lane 4, peptide: N-glycosidase F (PNGase). No difference in bandwidth or band density was observed between EndoH and PNGase F digestion, thus supporting lack of glycosylation of hGLUT1 expressed in yeast.
Fig. 2.
Fig. 2.
Electron density maps for hGLUT1–inhibitor complexes. Approximately equivalent views of the inhibitor binding sites (cytoplasmic side down) for three hGLUT1 complexes overlaid with 1σ likelihood-weighted 2Fo-Fc density, shown with blue contours. The complexes are plotted as sticks with the following color code: carbon, orange (protein) or green (inhibitor); oxygen, red; nitrogen, blue; bromine, dark red; and fluorine, cyan. Inhibitor-binding residues are labeled. Cytochalasin B (A), GLUT-i1 (B), and GLUT-i2 (C) are shown. This figure was prepared using PyMOL (The PyMOL Molecular Graphics System, Version 1.4.1; Schrödinger).
Fig. 3.
Fig. 3.
Interactions between inhibitors and hGLUT1. (Left) Schematics generated by LIGPLOT+ showing hGLUT1 residues that contact the inhibitors. The eyelash motif indicates a hydrophobic contact, and broken green lines indicate possible hydrogen bonds, with the hydrogen bond lengths shown in green. Residues that contact at least two of the three inhibitors are circled. (Right) Structures of the active sites of the complexes. The views are approximately the same as in Fig. 2. The inhibitors are shown as sticks. The protein is shown as semitransparent gold ribbons with side chains that contact the inhibitor shown as sticks using the following color code: carbon, gold (protein) or blue (inhibitor); oxygen, red; nitrogen, blue; bromine, dark red; and fluorine, green. Broken red lines denote hydrogen bonds. Cytochalasin B (A), GLUT-i1 (B), and GLUT-i2 (C) are shown. This figure was prepared using the UCSF Chimera package.
Fig. S2.
Fig. S2.
Unbiased electron density for the inhibitors in the three hGLUT1 complexes. The inhibitor-binding sites are shown as sticks: carbon, orange; oxygen, red; nitrogen, blue; fluorine, cyan. The bromine atom in GLUT-i2 is shown as a dark red sphere. Residues that contact the inhibitors are labeled. The views are the same as in Fig. 2. The likelihood-weighted difference density after refinement of the protein alone, before inhibitors were added to the structure, is shown with green contours, with contour levels of 2.1σ cytochalasin B (A) 2.4σ GLUT-i1 (B), and 2.5σ GLUT-i2 (C). This figure was prepared using the PyMOL Molecular Graphics System, version 1.4.1 (Schrödinger).
Fig. S3.
Fig. S3.
Density for hGLUT1–GLUT-i1 after refinement suggests partial occupancy of β-NG. (A) Likelihood-weighted 2Fo-Fc density (blue contours) and difference density (green contours, 3.5σ density; red contours, −3.5σ density) after refinement of hGLUT1–GLUT-i1. The complex is shown as sticks (carbon, magenta; oxygen, red; nitrogen, blue; fluorine, cyan). (B) mFo-DFc density is consistent with the oxygen substituents of the glucose moiety of a partially occupied β-NG molecule, shown modeled into the binding site identified in PDB ID code 4PYP as sticks with white carbons and red oxygens. However, there is no density for the nonyl chain of β-NG, and GLUT-i1 refines to almost full occupancy, so β-NG is most likely a minor component of the complex if it is present. This figure was prepared using the PyMOL Molecular Graphics System, version 1.4.1.
Fig. S4.
Fig. S4.
Cross-eyed stereo plots of (Fo1-Fo2)αcalc maps between all pairs of the inhibitor complexes. The views are the same as in Fig. 2. (A) Difference maps between hGLUT1–GLUT-i2 (Fo1) and hGLUT1–GLUT-i1 (Fo2), with αcalc from hGLUT1–GLUT-i2. Green contours indicate positive (2.7σ) density (density for hGLUT1–GLUT-i2 not present in hGLUT1–GLUT-i1), and red contours indicate negative (−2.7σ) density (density for hGLUT1–GLUT-i1 not present in hGLUT1–GLUT-i2). The two structures are overlaid on the map and shown as sticks (carbon, blue for hGLUT1–GLUT-i2 and cyan for hGLUT1–GLUT-i1; oxygen, red; nitrogen, blue; fluorine, cyan; bromine, dark red). (B) Difference maps between hGLUT1-cytochalasin B (Fo1) and hGLUT1–GLUT-i1 (Fo2), with αcalc from hGLUT1-cytochalasin B. Green and red contours indicate 3σ positive and negative density, respectively. The two structures are overlaid on the map and shown as sticks. hGLUT1-cytochalasin B has orange carbons, and hGLUT1–GLUT-i1 has cyan carbons. (C) Difference maps between hGLUT1-cytochalasin B (Fo1) and hGLUT1–GLUT-i2 (Fo2), with αcalc from hGLUT1-cytochalasin B. Green and red contours indicate 3σ positive and negative density, respectively. The two structures are overlaid on the map and shown as sticks. hGLUT1-cytochalasin B has orange carbons, and hGLUT1–GLUT-i1 has blue carbons. This figure was prepared using the PyMOL Molecular Graphics System, version 1.4.1.
Fig. 4.
Fig. 4.
Cytochalasin B docked into hGLUT1–4. The protein structures are homology models based on the crystal structures of hGLUT1 and hGLUT3, and are shown as ribbons with side chains that contact cytochalasin B, shown as sticks. Hydrogen bonds are denoted with broken yellow lines. The views are equivalent for all four proteins (cytoplasmic side down). (A) hGLUT1, color code: carbons, orange (protein); oxygen, red; nitrogen, blue. (B) hGLUT2, color code: carbons, magenta (protein); oxygen, red; nitrogen, blue. (C) hGLUT3 color code: carbons, teal; oxygen, red; nitrogen, blue. (D) hGLUT4, color code: carbons, green; oxygen, red; nitrogen, blue. Cytochalasin B is shown with yellow carbons for the four proteins.
Fig. 5.
Fig. 5.
GLUT-i1 docked into hGLUT1–4. The protein structures are homology models based on the crystal structures of hGLUT1 and hGLUT3, and are shown as ribbons with side chains that contact cytochalasin B, shown as sticks. Hydrogen bonds are denoted with broken yellow lines. The views are equivalent for all four proteins (cytoplasmic side down). (A) hGLUT1, color code: carbons, orange (protein); oxygen, red; nitrogen, blue. (B) hGLUT2, color code: carbons, magenta; oxygen, red; nitrogen, blue. (C) hGLUT3, color code: carbons, teal (protein); oxygen, red; nitrogen, blue. (D) hGLUT4, color code: carbons, green (protein); oxygen, red; nitrogen, blue. GLUT-i1 is shown with purple carbons for the four proteins with fluorine in cyan.
Fig. S5.
Fig. S5.
GLUT-i2 docked into hGLUT1–4. The protein structures are homology models based on the crystal structures of GLUT1 and GLUT3, and are shown as ribbons with side chains that contact GLUT-i2 shown as sticks. Hydrogen bonds are denoted with broken yellow lines. The views are equivalent for all four proteins (cytoplasmic side down). (A) hGLUT1, color code: carbons, orange (protein); oxygen, red; nitrogen, blue. (B) hGLUT2, color code: carbons, magenta (protein); oxygen, red; nitrogen, blue. (C) hGLUT3, color code: carbons, teal (protein); oxygen, red; nitrogen, blue. (D) hGLUT4, color code: carbons, green (protein); oxygen, red; nitrogen, blue. GLUT-i2 is shown with the following color code: carbons, olive green; oxygen, red; nitrogen, blue; and bromine, dark red, for the four proteins.
Fig. S6.
Fig. S6.
Overlapping inhibitor-binding sites in hGLUT1. The three hGLUT1-inhibitor structures were superposed, and the inhibitors were plotted as sticks with the following color code: Cytochalasin B, yellow; GLUT-i1, blue; GLUT-i2, red. The protein from hGLUT1–GLUT-i1 is plotted with ribbons, and the side chains that contact the inhibitor in all three complexes are shown as sticks and labeled. The C-terminal domain of hGLUT1 is pale blue, and the N-terminal domain is pale cyan. Helices are numbered with the conventional numbering for the major facilitator superfamily transporter fold. This figure was prepared using Chimera.

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