Fig 1.
ASCT2 models in different conformations reveal gating mechanism of the HP2 loop.
Side (A) and cytoplasmic (B) view of the ASCT2 models in the occluded conformation are represented in gray ribbons. The HP2 loop of the outward-open conformation (pink ribbons) is superposed to the occluded model. Atoms of the substrate cysteine are shown as spheres where oxygen atoms are displayed in red, sulfur in yellow, and carbon atoms in cyan. Sodium ions are illustrated as small purple spheres.
Fig 2.
Enrichment plots of the ASCT2 models indicate the suitability of the models for productive virtual screening.
Enrichment plots of ASCT2 in (A) the occluded conformation, (B) the outward open conformation and (C) the outward open conformation with Asp460 facing the binding site. The enrichment plots are represented in red, whereas the plot that is expected by random selection of ligands is represented in a dashed blue line. The bottom panel shows the enrichment in a semi-logarithmic scale.
Fig 3.
Binding sites of ASCT2 and GltPh differ in their shape, size, and polarity.
The final model of ASCT2 (gray) in an occluded conformation is superimposed on the X-ray structure of GltPh (pink). Key residues are displayed as lines, where oxygen and nitrogen atoms are colored in red and blue, respectively; the sodium ions are visualized as purple spheres. The L-aspartate coordinates from the GltPh structure are depicted by green sticks and L-serine coordinates derived from the docking of known ligands against the ASCT2 model are visualized with cyan sticks. (A) Hydrogen bonds between L-aspartate and GltPh are shown in dotted yellow lines and between L-serine and the ASCT2 model in dotted black lines. (B) The surface of the binding pocket is displayed in pink and gray for the template and the model, respectively, to visualize the additional pocket (pocket B) accessible in the model compared to the GltPh structure.
Fig 4.
Ligand binding mode for the ASCT2 occluded conformation model reveals key residues for substrate binding.
Predicted binding modes of selected known ligands (A-B) and ligands identified in this study (C-F). The backbone atoms of the ASCT2 binding site are visualized in gray cartoon; sidechain atoms of key residues are illustrated with gray lines and ligands are displayed as cyan sticks, with oxygen, nitrogen, and hydrogen atoms in red, blue, and white, respectively; hydrogen bonds between the small molecules and ASCT2 (involving residues Phe407, Gly448, Pro446, Ile445, Ser353, Thr468, Asn471) are displayed as dotted gray lines. The small molecule ligands are Threonine (A), 2-amino-3-(propionyloxy)propionic acid (B), aminooxetane-3-carboxylate (C), cis-3-hydroxyproline (D), acivicin (E), and L-DOPS (F).
Table 1.
Experimentally confirmed ligands.
Fig 5.
Electrophysiological methods confirm predicted activators and inhibitors.
(A-C) Representative whole-cell current traces in response to 1 mM of alanine, L-DOPS, and aminooxetane-3-carboxylate (AOC) applied at the time indicated by the gray bar. (D) Dose response curves for AOC and penicillamine (membrane potential = 0 mV, internal buffer contained 130 mM NaSCN and 10 mM alanine, external buffer contained 140 mM NaCl). (E) Maximum whole-cell currents relative to that induced by 1 mM alanine (membrane potential = 0 mV, internal buffer contained 130 mM NaSCN and 10 mM alanine, external buffer contained 140 mM NaCl).
Fig 6.
Outward-open conformation reveals an additional novel pocket.
(A) Predicted binding mode of the known inhibitor benzylcysteine derived from docking. The backbone atoms of ASCT2 are visualized as gray cartoon, with key residues establishing hydrogen bonds with the ligand in gray sticks, with oxygen, nitrogen, and hydrogen atoms in red, blue, and white, respectively. Two potential rotamers of Asp460 including the orientation facing the binding site (in pink) and surface (gray) are shown as sticks. Sodium ions are represented in purple spheres. (B) The binding site surface of the outward-open model of ASCT2 bound to benzylcysteine reveals the novel pocket (pocket A) resulting from the opening of HP2.
Fig 7.
Identification of a potent ASCT2 inhibitor based on the outward-open model.
(A) Predicted binding mode of the new identified inhibitor γ-FBP. The ASCT2 binding site is visualized as gray cartoon; sidechain atoms of key residues are illustrated with gray lines and the ligand is displayed as yellow sticks, with oxygen, nitrogen, and hydrogen atoms in red, blue, and white, respectively; hydrogen bonds between the inhibitor and ASCT2 (involving residues Ser351, Asp464, Thr468 and Asn471) are displayed as dotted black lines. (B) Representative current recordings in response to application of alanine, γ-FBP, and alanine + γ-FBP at conditions indicated in the legend. The gray bar depicts the duration of compound application. (C) Dose response relationship of γ-FBP-induced currents with the number of experiments averaged for each data point illustrated in brackets. (D) Alanine-induced currents (100 μM) in the presence of varying concentrations of γ-FBP (membrane potential = 0 mV, internal buffer contained 130 mM NaSCN and 10 mM alanine, external buffer contained 140 mM NaCl). The number of experiments averaged for each data point is illustrated in brackets. The dashed line represents the relationship based on a Ki of 87 μM, for comparison with (C).
Fig 8.
Chloroalanine, AOC and γ-FBP inhibit ASCT2-mediated glutamine uptake and cell viability in C8161 human melanoma cells.
(A-C) [3H]-L-glutamine uptake in C8161 cells was used to determine the IC50 of chloroalanine, AOC and γ-FBP (n = 3). (D-F) MTT cell viability assay (n = 3) in C8161 cells incubated with chloroalanine (5 mM), AOC (5 mM) and γ-FBP (5 mM). Two-way ANOVA test was performed to determine significance. (G-I) Apoptosis (Early, Annexin V+ PI-; Late, Annexin V+PI+) was examined by flow cytometry in C8161 cells incubated with chloroalanine (5 mM), AOC (5 mM) and γ-FBP (5 mM). Mann Whitney U test was used to determine significance. (J) ASCT2 expression (with GAPDH as a loading control) in C8161 cells was assessed after 48 hours incubation with chloroalanine (5 mM), AOC (5 mM) and γ-FBP (5 mM).