Browsing by keyword "facilitated diffusion"
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Small Molecule Modulation of GLUT1-Mediated Glucose TransportThe glucose transport protein, GLUT1, is highly expressed in rapidly proliferating cells, including cancer cells, while decreased GLUT1 levels are found in diseases such as GLUT1 deficiency syndrome and Alzheimer’s. There is increased interest in developing GLUT1 inhibitors as novel anticancer therapeutics, and the discovery of compounds that directly stimulate GLUT1 function. This work investigates how small molecules stimulate and/or inhibit GLUT1-mediated glucose transport, either directly or through the AMPK pathway. Using sugar transport assays and docking analyses to explore Ligand–GLUT1 interactions and specificity of binding, we show that: 1) Ligands inhibit GLUT1 by competing with glucose for binding to the exofacial or endofacial sugar binding sites; 2) Subsaturating inhibitor concentrations stimulate sugar uptake; 3) Ligands inhibit GLUT1–, GLUT3– and GLUT4–mediated sugar uptake in HEK293 cells; and 4) Inclusion of a benzonitrile head group on endofacial GLUT1 inhibitors confers greater inhibitory potency. Furthermore, we investigated AMPK-regulated GLUT1 trafficking in cultured blood-brain barrier endothelial cells, and show that inhibition of GLUT1 internalization is not responsible for increased cell surface levels of GLUT1 observed with AMPK activation in these cells. This study provides a framework for screening candidate GLUT1 inhibitors for specificity, and for optimizing drug design and delivery. Our data on transport stimulation at low inhibitor concentrations support the idea that GLUT1 functions as a cooperative oligomer of allosteric alternating access subunits.
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WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding SiteWZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 mum but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 mum but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 - GLUT1 - neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites.
