Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides

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.X-ray structure | glucose facilitator | human MFS transporter | cytochalasin B | GLUT inhibitor

Small‐Molecule Inhibition of Glucose Transporters GLUT‐1–4

“…GLUT isoform selectivity was determined in the same assay using DLD‐1 cells (express mainly GLUT‐1) and DLD‐1 GLUT1 (−/−) cells (express mainly GLUT‐3) or CHO cells that were stably transfected with hGLUT‐2 or hGLUT‐4. GLUT‐i1 and GLUT‐i2 preferably inhibit GLUT‐1 and GLUT‐4 over GLUT‐2 and GLUT‐3 (Table ) . GLUT‐i1 competes with glucose for the same binding site as determined in glucose competition experiments and crystal structure analysis with hGLUT‐1 (Table ) .…”

“…reported peptide analogues GLUT‐i1 and GLUT‐i2 (Figure ), with glucose uptake inhibitory activity. The cellular assay that was used monitored ATP depletion of hGLUT‐1‐ and luciferase‐transfected CHO‐K1 cells in the presence of a mitochondrial complex I inhibitor . GLUT‐i1 and GLUT‐i2 inhibit glycolytic ATP production with IC 50 values of 267 and 140 n m , respectively (Table ) .…”

“…GLUT‐i1 and GLUT‐i2 preferably inhibit GLUT‐1 and GLUT‐4 over GLUT‐2 and GLUT‐3 (Table ) . GLUT‐i1 competes with glucose for the same binding site as determined in glucose competition experiments and crystal structure analysis with hGLUT‐1 (Table ) . However, an impact of GLUT‐i1 or GLUT‐i2 on cancer cell growth has not been described.…”

Lack of appropriate stoichiometry: Strong evidence against an energetically important astrocyte–neuron lactate shuttle in brain

“…A clue that glycogen may be a source of lactate released in excess of glycolytic rate is that cytochalasin B (a glucose transport inhibitor [Jung and Rampal, ; Kapoor et al, ] that also has other effects, such as inhibition of actin polymerization (MacLean‐Fletcher and Pollard, ) was stated to inhibit ∼80% of [ 3 H]DG (and glucose) uptake, whereas this drug only inhibited basal or glutamate uptake–stimulated lactate‐pyruvate release by approximately 50% to 60% (see Methods and Fig. 4 in Pellerin and Magistretti, ).…”

Anticancer agents interacting with membrane glucose transporters

“…Therefore, interactions of inhibitors with this residue may hinder the movement of the loop that closes the cavity and, therefore, block the flux of glucose through the transporter. 80 …”