Enhanced Sampling Molecular Dynamics Simulations Reveal Transport Mechanism of Glycoconjugate Drugs through GLUT1.

Glucose transporters GLUT1 belong to the major facilitator superfamily and are essential to human glucose uptake. The overexpression of GLUT1 in tumor cells designates it as a pivotal target for glycoconjugate anticancer drugs. However, the interaction mechanism of glycoconjugate drugs with GLUT1 remains largely unknown. Here, we employed all-atom molecular dynamics simulations, coupled to steered and umbrella sampling techniques, to examine the thermodynamics governing the transport of glucose and two glycoconjugate drugs (i.e., 6-D-glucose-conjugated methane sulfonate and 6-D-glucose chlorambucil) by GLUT1. We characterized the specific interactions between GLUT1 and substrates at different transport stages, including substrate recognition, transport, and releasing, and identified the key residues involved in these procedures. Importantly, our results described, for the first time, the free energy profiles of GLUT1-transporting glycoconjugate drugs, and demonstrated that H160 and W388 served as important gates to regulate their transport via GLUT1. These findings provide novel atomic-scale insights for understanding the transport mechanism of GLUT1, facilitating the discovery and rational design of GLUT1-targeted anticancer drugs.

Rapid Enrichment of a Native Multipass Transmembrane Protein via Cell Membrane Electrophoresis through Buffer pH and Ionic Strength Adjustment.

Supported membrane electrophoresis is a promising technique for collecting membrane proteins in native bilayer environments. However, the slow mobility of typical transmembrane proteins has impeded the technique's advancement. Here, we successfully applied cell membrane electrophoresis to rapidly enrich a 12-transmembrane helix protein, glucose transporter 1 with antibodies (GLUT1 complex), by tuning the buffer pH and ionic strength. The identified conditions allowed the separation of the GLUT1 complex and a lipid probe, Fast-DiO, within a native-like environment in a few minutes. A force model was developed to account for distinct electric and drag forces acting on the transmembrane and aqueous-exposed portion of a transmembrane protein as well as the electroosmotic force. This model not only elucidates the impact of size and charge properties of transmembrane proteins but also highlights the influence of pH and ionic strength on the driving forces and, consequently, electrophoretic mobility. Model predictions align well with experimentally measured electrophoretic mobilities of the GLUT1 complex and Fast-DiO at various pH and ionic strengths as well as with several lipid probes, lipid-anchored proteins, and reconstituted membrane proteins from previous studies. Force analyses revealed the substantial membrane drag of the GLUT1 complex, significantly slowing down electrophoretic mobility. Besides, the counterbalance of similar magnitudes of electroosmotic and electric forces results in a small net driving force and, consequently, reduced mobility under typical neutral pH conditions. Our results further highlight how the size and charge properties of transmembrane proteins influence the suitable range of operating conditions for effective movement, providing potential applications for concentrating and isolating membrane proteins within this platform.

Identification of Structural Determinants of the Transport of the Dehydroascorbic Acid Mediated by Glucose Transport GLUT1.

GLUT1 is a facilitative glucose transporter that can transport oxidized vitamin C (i.e., dehydroascorbic acid) and complements the action of reduced vitamin C transporters. To identify the residues involved in human GLUT1's transport of dehydroascorbic acid, we performed docking studies in the 5 Å grid of the glucose-binding cavity of GLUT1. The interactions of the bicyclic hemiacetal form of dehydroascorbic acid with GLUT1 through hydrogen bonds with the -OH group of C3 and C5 were less favorable than the interactions with the sugars transported by GLUT1. The eight most relevant residues in such interactions (i.e., F26, Q161, I164, Q282, Y292, and W412) were mutated to alanine to perform functional studies for dehydroascorbic acid and the glucose analog, 2-deoxiglucose, in Xenopus laevis oocytes. All the mutants decreased the uptake of both substrates to less than 50%. The partial effect of the N317A mutant in transporting dehydroascorbic acid was associated with a 30% decrease in the Vmax compared to the wildtype GLUT1. The results show that both substrates share the eight residues studied in GLUT1, albeit with a differential contribution of N317. Our work, combining docking with functional studies, marks the first to identify structural determinants of oxidized vitamin C's transport via GLUT1.

GLUT3 inhibitor discovery through in silico ligand screening and in vivo validation in eukaryotic expression systems.

The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.

TXNIP interaction with GLUT1 depends on PI(4,5)P2.

GLUT1 is a major glucose facilitator expressed ubiquitously among tissues. Upregulation of its expression plays an important role in the development of many types of cancer and metabolic diseases. Thioredoxin-interacting protein (TXNIP) is an α-arrestin that acts as an adaptor for GLUT1 in clathrin-mediated endocytosis. It regulates cellular glucose uptake in response to both intracellular and extracellular signals via its control on GLUT1-4. In order to understand the interaction between GLUT1 and TXNIP, we generated GLUT1 lipid nanodiscs and carried out isothermal titration calorimetry and single-particle electron microscopy experiments. We found that GLUT1 lipid nanodiscs and TXNIP interact in a 1:1 ratio and that this interaction requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2).

Structural comparison of GLUT1 to GLUT3 reveal transport regulation mechanism in sugar porter family.

The human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here, we present a 2.4 Å crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic "SP motif" and a conserved "A motif" stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl- ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the SP family.

Target Score-A Proteomics Data Selection Tool Applied to Esophageal Cancer Identifies GLUT1-Sialyl Tn Glycoforms as Biomarkers of Cancer Aggressiveness.

Esophageal cancer (EC) is a life-threatening disease, demanding the discovery of new biomarkers and molecular targets for precision oncology. Aberrantly glycosylated proteins hold tremendous potential towards this objective. In the current study, a series of esophageal squamous cell carcinomas (ESCC) and EC-derived circulating tumor cells (CTCs) were screened by immunoassays for the sialyl-Tn (STn) antigen, a glycan rarely expressed in healthy tissues and widely observed in aggressive gastrointestinal cancers. An ESCC cell model was glycoengineered to express STn and characterized in relation to cell proliferation and invasion in vitro. STn was found to be widely present in ESCC (70% of tumors) and in CTCs in 20% of patients, being associated with general recurrence and reduced survival. Furthermore, STn expression in ESCC cells increased invasion in vitro, while reducing cancer cells proliferation. In parallel, an ESCC mass spectrometry-based proteomics dataset, obtained from the PRIDE database, was comprehensively interrogated for abnormally glycosylated proteins. Data integration with the Target Score, an algorithm developed in-house, pinpointed the glucose transporter type 1 (GLUT1) as a biomarker of poor prognosis. GLUT1-STn glycoproteoforms were latter identified in tumor tissues in patients facing worst prognosis. Furthermore, healthy human tissues analysis suggested that STn glycosylation provided cancer specificity to GLUT1. In conclusion, STn is a biomarker of worst prognosis in EC and GLUT1-STn glycoforms may be used to increase its specificity on the stratification and targeting of aggressive ESCC forms.

Orthosteric-allosteric dual inhibitors of PfHT1 as selective antimalarial agents.

Artemisinin-resistant malaria parasites have emerged and have been spreading, posing a significant public health challenge. Antimalarial drugs with novel mechanisms of action are therefore urgently needed. In this report, we exploit a "selective starvation" strategy by inhibiting Plasmodium falciparum hexose transporter 1 (PfHT1), the sole hexose transporter in P. falciparum, over human glucose transporter 1 (hGLUT1), providing an alternative approach to fight against multidrug-resistant malaria parasites. The crystal structure of hGLUT3, which shares 80% sequence similarity with hGLUT1, was resolved in complex with C3361, a moderate PfHT1-specific inhibitor, at 2.3-Å resolution. Structural comparison between the present hGLUT3-C3361 and our previously reported PfHT1-C3361 confirmed the unique inhibitor binding-induced pocket in PfHT1. We then designed small molecules to simultaneously block the orthosteric and allosteric pockets of PfHT1. Through extensive structure-activity relationship studies, the TH-PF series was identified to selectively inhibit PfHT1 over hGLUT1 and potent against multiple strains of the blood-stage P. falciparum Our findings shed light on the next-generation chemotherapeutics with a paradigm-shifting structure-based design strategy to simultaneously target the orthosteric and allosteric sites of a transporter.

In-vitro and in-vivo investigations into the carbene-gold anticancer drug candidates NHC*-Au-SCSNMe2 and NHC*-Au-S-GLUC against advanced prostate cancer PC3.

The anticancer drug candidates 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) dimethylamino dithiocarbamate and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-1-thiolate derivative exhibited nanomolar in-vitro activity against prostate cancer cells advanced prostate cancer (PC3) and micromolar inhibition of mammalian thioredoxin reductase. Encouraging maximum tolerable dose experiments led to human prostate cancer subcutaneous xenograft experiments; 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) dimethylamino dithiocarbamate and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-1-thiolate derivative were applied twelve times at two doses in groups of n = 5 PC3 to tumor-bearing NMRI:nu/nu mice. 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) dimethylamino dithiocarbamate and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-1-thiolate derivative at the dose of 10 and 20 mg/kg showed good tolerability, while no significant body weight loss was seen in both groups. In particular, for the drug 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) dimethylamino dithiocarbamate the tumor growth inhibition suggested to be dose dependent, reflected by the respective optimal T/C values of 0.45 at the dose of 10 mg/kg and of 0.31 at the dose of 20 mg/kg. By contrast, the 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-1-thiolate derivative treated groups showed no indication for dose-dependent antitumoral activity, as reflected by the optimal T/C values of 0.44 for the 10 mg/kg and for the 20 mg/kg treated mice. Immunohistochemical experiments involving Ki67 staining of tumor tissue showed that both compounds reduced PC3 cell proliferation against the difficult to treat advanced human prostate tumors derived from PC3.

In vitro gastrointestinal absorption of red wine anthocyanins - Impact of structural complexity and phase II metabolization.

Malvidin-3-O-glucoside, malvidin-3,5-O-diglucoside, malvidin-3-O-(6-O-coumaroyl)-glucoside-5-O-glucoside from Chinese Vitis davidii red wine were used to investigate the role of glucoside, diglucoside and coumaroylated glucoside moieties on their transport efficiency through MKN-28 gastric and Caco-2 intestinal cells. Due to the already described conversion of 3-O-glucosylated anthocyanins into 3-O-glucuronidated, the 3-O-glucuronidated metabolite of malvidin-3-O-glucoside was also tested. The antiproliferative activity was higher for the glucuronidated metabolite in both cell lines. All anthocyanins were transported through MKN-28 gastric cells and Caco-2 intestinal cells with transport efficiencies ranging from 4% to 9% in MKN-28 and from 3% to 5% in Caco-2. No significant differences on transport efficiencies were observed at 180 min among the different anthocyanins in MKN-28. The transport efficiency of malvidin-3-O-glucuronide at 180 min was about 3-4% in Caco-2 and MKN-28 cells. Computational studies were performed to evaluate the interaction between anthocyanins and glucose gastric transporters GLUT1 and GLUT3, which supported the experimental findings.

Mechanistic Insights into Protein Stability and Self-aggregation in GLUT1 Genetic Variants Causing GLUT1-Deficiency Syndrome.

Human sodium-independent glucose cotransporter 1 (hGLUT1) has been studied for its tetramerization and multimerization at the cell surface. Homozygous or compound heterozygous mutations in hGLUT1 elicit GLUT1-deficiency syndrome (GLUT1-DS), a metabolic disorder, which results in impaired glucose transport into the brain. The reduced cell surface expression or loss of function have been shown for some GLUT1 mutants. However, the mechanism by which deleterious mutations affect protein structure, conformational stability and GLUT1 oligomerization is not known and require investigation. In this review, we combined previous knowledge of GLUT1 mutations with hGLUT1 crystal structure to analyze native interactions and several natural single-point mutations. The modeling of native hGLUT1 structure confirmed the roles of native residues in forming a range of side-chain interactions. Interestingly, the modeled mutants pointed to the formation of a variety of non-native novel interactions, altering interaction networks and potentially eliciting protein misfolding. Self-aggregation of the last part of hGLUT1 was predicted using protein aggregation prediction tool. Furthermore, an increase in aggregation potential in the aggregation-prone regions was estimated for several mutants suggesting increased aggregation of misfolded protein. Protein stability change analysis predicted that GLUT1 mutant proteins are unstable. Combining GLUT1 oligomerization behavior with our modeling, aggregation prediction, and protein stability analyses, this work provides state-of-the-art view of GLUT1 genetic mutations that could destabilize native interactions, generate novel interactions, trigger protein misfolding, and enhance protein aggregation in a disease state.

A bio-inspired highly selective enzymatic glucose sensor using a red blood cell membrane.

In the development of enzymatic glucose sensors, accurate glucose sensing has been a challenging task because of the existence of numerous interfering molecules in the blood. Meanwhile, red blood cells (RBCs) selectively uptake glucose via a membrane protein called glucose transporter-1. In this study, we developed the RBC membrane (RBCM)-coated enzymatic glucose sensors that mimic the glucose uptake. The RBCM-coated sensors were examined via scanning electron microscopy, atomic force microscopy, and ATR-FTIR. We optimized the glucose permeability of the RBCM filter by controlling the thickness of the filter. The sensing range of the optimized sensor was 1-15 mM, the detection limit was 0.66 mM, and the sensitivity was 2.978 µA mM-1. Intriguingly, the RBCM-coated sensor was highly accurate and precise, despite the coexistence of glucose and interfering molecules (e.g., mannose, galactose, ascorbic acid, uric acid, and cysteine). For each interfering molecule, the errors of our sensor were 0.8 to 2.3%, which was 4.8-14.2 times more accurate than the uncoated one. A similar result was verified for a human serum sample containing countless interfering molecules. Also, the sensing performance of the sensor was consistent after 4 weeks of storage. The results suggest that applying RBCM may improve the selectivity of various types of glucose sensors including the continuous monitoring system.

Small-Molecule Inhibition of Glucose Transporters GLUT-1-4.

Glucose addiction is observed in cancer and other diseases that are associated with hyperproliferation. The development of compounds that restrict glucose supply and decrease glycolysis has great potential for the development of new therapeutic approaches. Addressing facilitative glucose transporters (GLUTs), which are often upregulated in glucose-dependent cells, is therefore of particular interest. This article reviews a selection of potent, isoform-selective GLUT inhibitors and their biological characterization. Potential therapeutic applications of GLUT inhibitors in oncology and other diseases that are linked to glucose addiction are discussed.

Pharmacological Targeting of GLUT1 to Control Autoreactive T Cell Responses.

An increasing body of evidence indicates that bio-energetic metabolism of T cells can be manipulated to control T cell responses. This potentially finds a field of application in the control of the T cell responses in autoimmune diseases, including in type 1 diabetes (T1D). Of the possible metabolic targets, Glut1 gained considerable interest because of its pivotal role in glucose uptake to fuel glycolysis in activated T cells, and the recent development of a novel class of small molecules that act as selective inhibitor of Glut1. We believe we can foresee a possible application of pharmacological Glut1 blockade approach to control autoreactive T cells that destroy insulin producing beta cells. However, Glut1 is expressed in a broad range of cells in the body and off-target and side effect are possible complications. Moreover, the duration of the treatment and the age of patients are critical aspects that need to be addressed to reduce toxicity. In this paper, we will review recent literature to determine whether it is possible to design a pharmacological Glut1 blocking strategy and how to apply this to autoimmunity in T1D.

Conformational Studies of Glucose Transporter 1 (GLUT1) as an Anticancer Drug Target.

Glucose transporter 1 (GLUT1) is a facilitative glucose transporter overexpressed in various types of tumors; thus, it has been considered as an important target for cancer therapy. GLUT1 works through conformational switching from an outward-open (OOP) to an inward-open (IOP) conformation passing through an occluded conformation. It is critical to determine which conformation is preferred by bound ligands because the success of structure-based drug design depends on the appropriate starting conformation of the target protein. To find out the most favorable GLUT 1 conformation for ligand binding, we ran systemic molecular docking studies for different conformations of GLUT1 using known GLUT1 inhibitors. Our data revealed that the IOP is the preferred conformation and that residues Phe291, Phe379, Glu380, Trp388, and Trp412 may play critical roles in ligand binding to GLUT1. Our data suggests that conformational differences in these five amino acids in the different conformers of GLUT1 may be used to design ligands that inhibit GLUT1.

New insights into GluT1 mechanics during glucose transfer.

Glucose plays a crucial role in the mammalian cell metabolism. In the erythrocytes and endothelial cells of the blood-brain barrier, glucose uptake is mediated by the glucose transporter type 1 (GluT1). GluT1 deficiency or mutations cause severe physiological disorders. GluT1 is also an important target in cancer therapy as it is overexpressed in tumor cells. Previous studies have suggested that GluT1 mediates solute transfer through a cycle of conformational changes. However, the corresponding 3D structures adopted by the transporter during the transfer process remain elusive. In the present work, we first elucidate the whole conformational landscape of GluT1 in the absence of glucose, using long molecular dynamics simulations and show that the transitions can be accomplished through thermal fluctuations. Importantly, we highlight a strong coupling between intracellular and extracellular domains of the protein that contributes to the transmembrane helices reorientation during the transition. The conformations adopted during the simulations differ from the known 3D bacterial homologs structures resolved in similar states. In holo state simulations, we find that glucose transits along the pathway through significant rotational motions, while maintaining hydrogen bonds with the protein. These persistent motions affect side chains orientation, which impacts protein mechanics and allows glucose progression.

Ascorbic acid-modified brain-specific liposomes drug delivery system with "lock-in" function.

In this study, a novel brain targeting ascorbic acid (AA) derivative with "lock-in" function was designed and synthesized as a liposome ligand to prepare novel liposomes to achieve the effective delivery of drug formulations to brain via glucose transporter 1 (GLUT1) and the Na+-dependent vitamin C transporter (SVCT2). The liposome was prepared and characterized in terms of the particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis and cell cytotoxicity. The preliminary evaluation in vivo demonstrated that the AA-thiamine disulfide system (TDS)-coated liposome had an improved targeting ability and significantly increased the brain concentration of docetaxel (DTX) as compared to the naked docetaxel, the non-coated and the AA-coated liposomes. The relative uptake efficiency and concentration efficiency were enhanced by 3.24- and 5.62-fold compared to that of the naked docetaxel, respectively. Both distribution data and pharmacokinetic parameters suggested that the ascorbic acid thiamine disulfide delivery system was a promising carrier to enhance central nervous system (CNS) drug's delivery ability into brain.

Molecular docking of anti-inflammatory drug diclofenac with metabolic targets: Potential applications in cancer therapeutics.

Diclofenac is a potent NSAID of clinical choice, which is widely used for containing inflammation. Moreover, recent experimental evidences overwhelmingly substantiate its antineoplastic potential. However, the precise molecular mechanisms of diclofenac's anticancer activity remain poorly understood. Neoplastic cells display reprogrammed metabolic features, which are manifested and regulated by a complex networking of molecular pathways. However, the effect of diclofenac on tumor cell metabolism are not yet clearly deciphered. Hence, the present investigation was carried out to identify and characterize key diclofenac targets of tumor metabolism, cell survival and chemoresistance. The interactions of diclofenac with such targets was analysed by PatchDock and YASARA (Yet Another Scientific Artificial Reality Application). The docking ability of diclofenac with its targets was based on analysis of dissociation constant (Kd), geometric shape complementarity score (GSC score), approximate interface area (AI area) and binding energy. The findings of this investigation reveal that diclofenac is capable of interacting with all of the selected molecular targets. Prominent interactions were observed with GLUT1, MCT4, LDH A, COX1, COX2, BCRP/ABCG2, HDM2/MDM2 and MRP1 compared to other targets. Interactions were of noncovalent nature involving ionic, hydrophobic interactions, Van der Waals forces and H-bonds, which varied depending on targets. This study for the first time, characterizes the nature of molecular interactions of diclofenac with selected targets involved in cancer cell metabolism, pH homeostasis, chemosensitivity, cell signalling and inflammation. Hence, these findings will be highly beneficial in optimizing the utility of diclofenac in development of novel cancer therapeutics.

Methylation and Acetylation Enhanced the Antidiabetic Activity of Some Selected Flavonoids: In Vitro, Molecular Modelling and Structure Activity Relationship-Based Study.

Flavonoids have been reported to exert antihyperglycemic effects and have potential to enhance the current therapy options against type 2 diabetes mellitus. However, the structure activity relationships (SAR) studies of flavonoids against this disease have not been thoroughly comprehended. Hence, in the present study, 14 structurally related flavonoids viz. wogonin, techtochrysin, norwogonin, isoscutellarein, hypolaetin, kaempferol, quercetin, methyl ether of wogonin, acetate of wogonin, acetate of norwogonin, 8-hydroxy-7-methoxyflavone, chrysin, (+)-catechin and (-)-epicatechin were taken into account for in vitro antidiabetic evaluation. Cell viability of RIN-5F pancreatic cells and 3T3-L1 pre-adipocyte cells was initially tested, then an insulin secretion assay of RIN-5F as well as adipogenesis and glucose uptake measurements of adipocyte were investigated. Subsequently, protein expressions study through adipokines measurement (leptin, adiponectin, TNF-α, RBP-4) via enzyme-linked immunosorbent assay (ELISA) kit, Western blotting analysis against GLUT4 and C/EBP-α as well as molecular docking against GLUT1 were analyzed. The results from cell culture antidiabetic assays (insulin secretion, adipogenesis, and glucose uptake), protein expressions and molecular docking pointed that the methoxy group at position C-8 is responsible for antidiabetic property of selected flavonoids via glucose uptake mechanism indicated by up regulation of GLUT4 and C/EBP-α expressions. The mechanism could be enhanced by the addition of an acetate group at C-5 and C-7 of the flavone skeleton.

Red wine and green tea flavonoids are cis-allosteric activators and competitive inhibitors of glucose transporter 1 (GLUT1)-mediated sugar uptake.

The antioxidant- and flavonoid-rich contents of red wine and green tea are reported to offer protection against cancer, cardiovascular disease, and diabetes. Some studies, however, show that flavonoids inhibit GLUT1-mediated, facilitative glucose transport, raising the possibility that their interaction with GLUT1 and subsequent downstream effects on carbohydrate metabolism may also impact health. The present study explores the structure-function relationships of flavonoid-GLUT1 interactions. We find that low concentrations of flavonoids act as cis-allosteric activators of sugar uptake, whereas higher concentrations competitively inhibit sugar uptake and noncompetitively inhibit sugar exit. Studies with heterologously expressed human GLUT1, -3, or -4 reveal that quercetin-GLUT1 and -GLUT4 interactions are stronger than quercetin-GLUT3 interactions, that epicatechin gallate (ECG) is more selective for GLUT1, and that epigallocatechin gallate (EGCG) is less GLUT isoform-selective. Docking studies suggest that only one flavonoid can bind to GLUT1 at any instant, but sugar transport and ligand-binding studies indicate that human erythrocyte GLUT1 can bind at least two flavonoid molecules simultaneously. Quercetin and EGCG are each characterized by positive, cooperative binding, whereas ECG shows negative cooperative binding. These findings support recent studies suggesting that GLUT1 forms an oligomeric complex of interacting, allosteric, alternating access transporters. We discuss how modulation of facilitative glucose transporters could contribute to the protective actions of the flavonoids against diabetes and Alzheimer's disease.