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.

Identification of new GLUT2-selective inhibitors through in silico ligand screening and validation in eukaryotic expression systems.

Glucose is an essential energy source for cells. In humans, its passive diffusion through the cell membrane is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT2 transports both glucose and fructose with low affinity and plays a critical role in glucose sensing mechanisms. Alterations in the function or expression of GLUT2 are involved in the Fanconi-Bickel syndrome, diabetes, and cancer. Distinguishing GLUT2 transport in tissues where other GLUTs coexist is challenging due to the low affinity of GLUT2 for glucose and fructose and the scarcity of GLUT-specific modulators. By combining in silico ligand screening of an inward-facing conformation model of GLUT2 and glucose uptake assays in a hexose transporter-deficient yeast strain, in which the GLUT1-5 can be expressed individually, we identified eleven new GLUT2 inhibitors (IC50 ranging from 0.61 to 19.3 µM). Among them, nine were GLUT2-selective, one inhibited GLUT1-4 (pan-Class I GLUT inhibitor), and another inhibited GLUT5 only. All these inhibitors dock to the substrate cavity periphery, close to the large cytosolic loop connecting the two transporter halves, outside the substrate-binding site. The GLUT2 inhibitors described here have various applications; GLUT2-specific inhibitors can serve as tools to examine the pathophysiological role of GLUT2 relative to other GLUTs, the pan-Class I GLUT inhibitor can block glucose entry in cancer cells, and the GLUT2/GLUT5 inhibitor can reduce the intestinal absorption of fructose to combat the harmful effects of a high-fructose diet.

Blockade of fructose transporter protein GLUT5 inhibits proliferation of colon cancer cells: proof of concept for a new class of anti-tumor therapeutics.

BACKGROUND: Despite the fact that colorectal cancer (CRC) is one of the most commonly diagnosed cancers in men and women, its current treatment remains unsatisfactory and therefore novel studies proposing new approaches are necessary. A high sugar diet is believed to promote carcinogenesis. Fructose is absorbed from the gastrointestinal tract by members of the glucose transporter family-GLUT. The aim of the study was to characterize the expression of GLUT5 at mRNA level in CRC patients. Moreover, our goal was to elucidate the molecular role of GLUT5 in CRC and assess whether GLUT5 inhibitor may affect the viability of colon cancer cells. METHODS: The expression of GLUT5 at mRNA level was characterized based on 30 samples from resected colorectal cancers and 30 healthy colonic mucosa specimens from surgical margins. The inhibitory effect of N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSBNA) was assessed on a colon cancer cell line, HT-29, and normal colon epithelium cells-CCD 841 CoN Cells. RESULTS: GLUT5 expression was found in 96.7% of cancer specimens and only in 53.3% of healthy mucosa fragments. In cancer tissue, real-time PCR analysis showed almost 2, fivefold (p< 0.001) increase of GLUT5 mRNA expression level compared with the healthy intestinal mucosa. GLUT5 inhibitor, MSNBA (10 µM) significantly decreased the viability of colon cancer cells, while barely affected the viability of normal colon epithelium cells. CONCLUSIONS: Our study suggests that a strong focus should be put on GLUT5 and its inhibitors for both diagnostic and therapeutic purposes in CRC.

Differential patterns of inhibition of the sugar transporters GLUT2, GLUT5 and GLUT7 by flavonoids.

Only limited data are available on the inhibition of the sugar transporter GLUT5 by flavonoids or other classes of bioactives. Intestinal GLUT7 is poorly characterised and no information exists concerning its inhibition. We aimed to study the expression of GLUT7 in Caco-2/TC7 intestinal cells, and evaluate inhibition of glucose transport by GLUT2 and GLUT7, and of fructose transport by GLUT2, GLUT5 and GLUT7, by flavonoids. Differentiated Caco-2/TC7 cell monolayers were used to investigate GLUT7 expression, as well as biotinylation and immunofluorescence to assess GLUT7 location. For mechanistic sugar transport studies, X. laevis oocytes were injected with individual mRNA, and GLUT protein expression on oocyte membranes was confirmed. Oocytes were incubated with D-[14C(U)]-glucose or D-[14C(U)]-fructose in the presence of flavonoids, and uptake was estimated by liquid scintilation counting. In differentiated Caco-2/TC7 cell monolayers, GLUT7 was mostly expressed apically. When applied apically, or to both compartments, sorbitol, galactose, L-glucose or sucrose did not affect GLUT7 mRNA expression. Fructose applied to both sides increased GLUT7 mRNA (13%, p ≤ 0.001) and total GLUT7 protein (2.7-fold, p ≤ 0.05), while the ratio between apical, basolateral and total GLUT7 protein was unchanged. In the X. laevis oocyte model, GLUT2-mediated glucose and fructose transport were inhibited by quercetin, (-)-epigallocatechin gallate (EGCG) and apigenin, GLUT5-mediated fructose transport was inhibited by apigenin and EGCG, but not by quercetin, and GLUT7-mediated uptake of both glucose and fructose was inhibited by apigenin, but not by quercetin nor EGCG. Expression of GLUT7 was increased by fructose, but only when applied to Caco-2/TC7 cells both apically and basolaterally. Since GLUT2, GLUT5 and GLUT7 show different patterns of inhibition by the tested flavonoids, we suggest that they have the potential to be used as investigational tools to distinguish sugar transporter activity in different biological settings.

Green and Chamomile Teas, but not Acarbose, Attenuate Glucose and Fructose Transport via Inhibition of GLUT2 and GLUT5.

SCOPE: High glycaemic sugars result in blood-glucose spikes, while large doses of post-prandial fructose inundate the liver, causing an imbalance in energy metabolism, both leading to increased risk of metabolic malfunction and type 2 diabetes. Acarbose, used for diabetes management, reduces post-prandial hyperglycaemia by delaying carbohydrate digestion. METHODS AND RESULTS: Chamomile and green teas both inhibited digestive enzymes (α-amylase and maltase) related to intestinal sugar release, as already established for acarbose. However, acarbose had no effect on uptake of sugars using both differentiated human Caco-2 cell monolayers and Xenopus oocytes expressing human glucose transporter-2 (GLUT2) and GLUT5. Both teas effectively inhibited transport of fructose and glucose through GLUT2 inhibition, while chamomile tea also inhibited GLUT5. Long term incubation of Caco-2/TC7 cells with chamomile tea for 16 h or 4 days did not enhance the observed effects, indicating that inhibition is acute. Sucrase activity was directly inhibited by green tea and acarbose, but not chamomile. CONCLUSION: These findings show that chamomile and green teas are potential tools to manage absorption and metabolism of sugars with efficacy against high sugar bolus stress inflicted, for example, by high fructose syrups, where the drug acarbose would be ineffective.

Establishing a yeast-based screening system for discovery of human GLUT5 inhibitors and activators.

Human GLUT5 is a fructose-specific transporter in the glucose transporter family (GLUT, SLC2 gene family). Its substrate-specificity and tissue-specific expression make it a promising target for treatment of diabetes, metabolic syndrome and cancer, but few GLUT5 inhibitors are known. To identify and characterize potential GLUT5 ligands, we developed a whole-cell system based on a yeast strain deficient in fructose uptake, in which GLUT5 transport activity is associated with cell growth in fructose-based media or assayed by fructose uptake in whole cells. The former method is convenient for high-throughput screening of potential GLUT5 inhibitors and activators, while the latter enables detailed kinetic characterization of identified GLUT5 ligands. We show that functional expression of GLUT5 in yeast requires mutations at specific positions of the transporter sequence. The mutated proteins exhibit kinetic properties similar to the wild-type transporter and are inhibited by established GLUT5 inhibitors N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSNBA) and (-)-epicatechin-gallate (ECG). Thus, this system has the potential to greatly accelerate the discovery of compounds that modulate the fructose transport activity of GLUT5.

Discovery of a specific inhibitor of human GLUT5 by virtual screening and in vitro transport evaluation.

GLUT5, a fructose-transporting member of the facilitative glucose transporter (GLUT, SLC2) family, is a therapeutic target for diabetes and cancer but has no potent inhibitors. We virtually screened a library of 6 million chemicals onto a GLUT5 model and identified N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSNBA) as an inhibitor of GLUT5 fructose transport in proteoliposomes. MSNBA inhibition was specific to GLUT5; this inhibitor did not affect the fructose transport of human GLUT2 or the glucose transport of human GLUT1-4 or bacterial GlcPSe. In MCF7 cells, a human breast cancer cell line, MSNBA competitively inhibited GLUT5 fructose uptake with a KI of 3.2 ± 0.4 µM. Ligand docking, mutagenesis and functional studies indicate that MSNBA binds near the active site and inhibitor discrimination involves H387 of GLUT5. Thus, MSNBA is a selective and potent inhibitor of fructose transport via GLUT5, and the first chemical probe for this transporter. Our data indicate that active site differences in GLUT members could be exploited to further enhance ligand specificity.

Inhibition of human GLUT1 and GLUT5 by plant carbohydrate products; insights into transport specificity.

Glucose transporters GLUT1 (transports glucose) and GLUT5 (transports fructose), in addition to their functions in normal metabolism, have been implicated in several diseases including cancer and diabetes. While GLUT1 has several inhibitors, none have been described for GLUT5. By transport activity assays we found two plant products, rubusoside (from Rubus suavissimus) and astragalin-6-glucoside (a glycosylated derivative of astragalin, from Phytolacca americana) that inhibited human GLUT5. These plants are utilized in traditional medicine: R. suavissimus for weight loss and P. americana for cancer treatment, but the molecular interactions of these products are unknown. Rubusoside also inhibited human GLUT1, but astragalin-6-glucoside did not. In silico analysis of rubusoside:protein interactions pinpointed a major difference in substrate cavity between these transporters, a residue that is a tryptophan in GLUT1 but an alanine in GLUT5. Investigation of mutant proteins supported the importance of this position in ligand specificity. GLUT1W388A became susceptible to inhibition by astragalin-6-glucoside and resistant to rubusoside. GLUT5A396W transported fructose and also glucose, and maintained inhibition by rubusoside and astragalin-6-glucoside. Astragalin-6-glucoside can serve as a starting point in the design of specific inhibitors for GLUT5. The application of these studies to understanding glucose transporters and their interaction with substrates and ligands is discussed.

Synthesis and characterization of 6-deoxy-6-fluoro-D-fructose as a potential compound for imaging breast cancer with PET.

FDG-based imaging with positron emission tomography (PET) has been widely used in the detection of cancer, but has not reached its full potential. In breast cancer, the glucose/fructose transporter GLUT2 and the fructose transporter GLUT5 are known to be overexpressed in transformed tissues, implicating that a fructose-based analogue would be a useful target for the improved imaging of breast cancer. We have successfully synthesized the fluorinated fructose compound, 6-deoxy-6-fluoro-D-fructose (6FDF) and examined its potential for transport and accumulation in breast cancer cells. Expression analysis of GLUT isoforms was performed on two GLUT5 expressing breast cancer cell lines using western blotting and immunocytochemistry. Uptake and inhibition studies were undertaken using [14C]-labelled hexoses. Transport inhibition studies showed dose dependent inhibition of fructose transport in both cell lines by the newly synthesized 6-deoxy-6-fluoro-D-fructose (6FDF). Also, near linear uptake over time of [14C]-labelled 6FDF was observed in both cell lines. It appears that 6FDF may have great promise for use in in vivo PET imaging of breast cancer. Ongoing work will confirm the efficacy of this compound in imaging in mouse models.