Glucose transporter 1 (Glut1)
WZB117 reduced glucose transport in cancer cells in a dose-dependent manner, according to the glucose uptake assay. The assay was completed in less than a minute, indicating that WZB117-induced reduction of glucose transport may be the result of a quick and direct mechanism. WZB117 reduced cancer cell proliferation with an IC50 of about 10 μM, according to an experiment for cell viability. The clonogenic experiment verified WZB117's inhibitory effect on cancer cell development and demonstrated the irreversible nature of this inhibition. WZB117 therapy had a far greater cell growth inhibitory effect on lung cancer A549 cells than it did on non-tumorigenic lung NL20 cells. MCF7 breast cancer cells and their non-tumorigenic MCF12A counterparts showed comparable outcomes. Greater suppression of cell development was reported when WZB117 was given to cancer cells cultivated under hypoxia settings as opposed to normoxic ones [1].

WZB117 reduced glucose transport in cancer cells in a dose-dependent manner, according to the glucose uptake assay. The assay was completed in less than a minute, indicating that WZB117-induced reduction of glucose transport may be the result of a quick and direct mechanism. WZB117 reduced cancer cell proliferation with an IC50 of about 10 μM, according to an experiment for cell viability. The clonogenic experiment verified WZB117's inhibitory effect on cancer cell development and demonstrated the irreversible nature of this inhibition. WZB117 therapy had a far greater cell growth inhibitory effect on lung cancer A549 cells than it did on non-tumorigenic lung NL20 cells. MCF7 breast cancer cells and their non-tumorigenic MCF12A counterparts showed comparable outcomes. Greater suppression of cell development was reported when WZB117 was given to cancer cells cultivated under hypoxia settings as opposed to normoxic ones [1].

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WZB117 inhibited glucose transport in human non-small cell lung cancer (NSCLC) A549 cells in a dose-dependent manner. [1]
WZB117 (30 μmol/L) rapidly and completely inhibited glucose transport within 1 minute in A549 cells. [1]
WZB117 inhibited cancer cell proliferation with an IC50 of approximately 10 μmol/L in A549 cells as measured by MTT assay. [1]
Clonogenic assays showed that WZB117 treatment led to irreversible cell growth inhibition in A549, H1299, and MCF7 cancer cell lines. [1]
WZB117 inhibited cell proliferation in human lung cancer A549 cells significantly more than in non-tumorigenic lung NL20 cells at 48 hours post-treatment. A similar selective effect was observed in breast cancer MCF7 cells compared to non-tumorigenic MCF12A cells. [1]
WZB117 treatment under hypoxic conditions resulted in greater cell growth inhibition in A549 cells compared to normoxic conditions. [1]
Synergistic anticancer effects were observed between WZB117 and the chemotherapeutic drugs cisplatin or paclitaxel. [1]
WZB117 (30 μmol/L) inhibited glucose transport in human red blood cells (RBCs), which express Glut1 as their sole glucose transporter. [1]
WZB117 also inhibited glucose transport in RBC-derived inside-out vesicles (IOV) and right-side-out vesicles (ROV). [1]
In A549 cells, WZB117 treatment (10 μmol/L) led to a decrease in Glut1 protein levels as early as 12 hours, while Glut1 mRNA levels were upregulated at 24 hours. [1]
WZB117 treatment reduced extracellular lactate levels and intracellular ATP levels in A549 cells as early as 6-12 hours, with further decline at 24 hours. [1]
Addition of extracellular ATP (0.5-10 mmol/L) at the time of WZB117 (30 μmol/L) addition significantly increased intracellular ATP and rescued A549 cell viability after 24 hours. Delayed ATP addition (after 12 hours) lost its rescue ability. ATP addition did not rescue cells treated with paclitaxel. [1]
WZB117 treatment led to autophagy as early as 6 hours post-treatment. [1]
Co-treatment of A549 cells with WZB117 (1 μmol/L) and the mitochondrial inhibitor oligomycin (50 nmol/L) resulted in further reduction of cell proliferation compared to WZB117 alone. [1]
WZB117 treatment reduced levels of key glycolytic enzymes hexokinase II and PKM2 at 6 and/or 12 hours, but they were upregulated at 24 hours. PGAM1 levels were not affected. [1]
Western blot analysis showed that WZB117 treatment decreased phosphorylated Akt and mTOR levels at 6 and 12 hours, and upregulated phosphorylated AMPK levels coinciding with ATP decline. [1]
WZB117 induced endoplasmic reticulum stress, indicated by increased GRP78/BiP protein levels from 6 to 48 hours and increased phosphorylated eIF2α, but did not significantly increase CHOP expression or induce substantial apoptosis (low PARP cleavage). [1]
Flow cytometric analysis showed WZB117 treatment resulted in approximately 8% increase in necrosis and only about 2% apoptosis in A549 cells at 48 hours. [1]
WZB117 treatment caused cell-cycle arrest in A549 cells, with approximately 23% more cells in G0-G1 phase, 4% more in G2-M phase, and 30% fewer cells in S phase at 24 hours. This was accompanied by decreased levels of CDK2, cyclin E2, and phosphorylated retinoblastoma (pRb). [1]
WZB117 treatment induced senescence in A549 cells at 24 hours, as evidenced by enlarged cell morphology, expression of senescence-associated β-galactosidase (β-gal), irreversible growth inhibition, and changes in pRb. [1]

Compared to mock (PBS/DMSO)-treated tumors, compound-treated tumors had an average size reduction of almost 70% following daily intraperitoneal injection of WZB117 at a dose of 10 mg/kg body weight, according to animal studies. Most astonishingly, after treatment, 2 out of 10 tumors treated with compounds stopped growing and even vanished during the trial. Compared to mock-treated mice, WZB117-treated mice lost between 1 and 2 grams of body weight, with adipose tissue accounting for the majority of the weight loss. These findings were based on measurements and analysis of body weight. The study's conclusion revealed that while cell counts stayed within normal ranges, the compound-treated mice's lymphocyte and platelet counts differed from the vehicle-treated mice's. Use of glucose transport inhibitors raises certain concerns since treated mice may develop hyperglycemia [1].

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Daily intraperitoneal injection of WZB117 at 10 mg/kg body weight for 10 weeks resulted in a more than 70% reduction in the average tumor volume of human A549 lung cancer xenografts in nude mice compared to the PBS/DMSO vehicle control group. Notably, 2 out of 10 tumors in the treatment group disappeared during the study. [1]

Protein target studies I: RBC membrane vesicle preparation and glucose uptake assay[1]
RBC and RBC-derived vesicles were prepared using published protocols with minor modifications. The glucose uptake assay using sealed vesicles was similar to that in RBCs, except that the centrifugation was at 18,000 × g for 20 minutes to precipitate the vesicles after each washing step.

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Protein target studies II: docking studies[1]
A molecular model of WZB117 was constructed using Spartan 10. Following molecular mechanics energy minimization with the Merck molecular force field, the compound structures were exported to Macromodel and docked to the Glut1 homology modeled PDB structure 1SUK Protein and grid preparations were conducted using the Glide module of FirstDiscovery 2.7 with default protocols and centered in the middle of the transport channel with the bounding box encompassing the entire channel. WZB117 was then docked using Glide, and the best docked structure for the compound was selected on the basis of the Glide-calculated Emodel value.

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Western blot analyses and RNA isolation and real-time PCR[1]
Western blot analyses were conducted using the standard protocol. Antibodies for Glut1 (H-43), eIF2α, and cyclophosphamide–Adriamycin–vincristine–prednisone (CHOP) were from Santa Cruz; PGAM1 antibody was from Novus Biologicals. Antibody for p-eIF2α was from Invitrogen.

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RNA from treated A549 cells was isolated using RNeasy total RNA extraction kit, and cDNA was synthesized with the Bio-Rad iScript Select cDNA Synthesis Kit. The produced cDNA was used to specifically quantify the transcript of SLC2A1 (Glut1) using the Bio-Rad iCycler with the Bio-Rad iQ SyBr Green Supermix Kit. The RT2-PCR primer sets for human SLC2A1 and β-actin were from SuperArray. For quantifying transcript levels, δCt method was used. β-actin mRNA was used as an internal control for normalizing Glut1 mRNA.

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Lactate and ATP measurements and ATP rescue study[1]
Extracellular lactate concentration was measured using the Lactate Assay Kit II. Intracellular ATP concentration was measured using ATPlite luminescence ATP detection assay system from Perkin-Elmer. Briefly, cells were seeded at a density of 50,000 cells in each well of a 96-well plate. ATP levels were measured after 6, 12, and 24 hours of treatment. Protein concentration of cells in each well was determined for both lactate and ATP measurements for signal normalization. In the cell rescue study, ATP of various concentrations were added in cell culture medium of cancer cells in 96-well plates with or without 30 μmol/L WZB117. Intracellular ATP levels and cell viability were measured by an MTT assay 24 hours after the treatment.

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Molecular docking studies were conducted to investigate the interaction between WZB117 and Glut1. A homology model of human Glut1 (based on PDB structure 1SUK) was used. The compound structure was energy-minimized and then docked into the Glut1 transport channel using a grid-based docking method. The docking was centered in the middle of the channel with a bounding box encompassing the entire channel. The best docked structure was selected based on the calculated Glide score (Emodel value). The analysis suggested that WZB117 binds to the central channel region of Glut1, forming three hydrogen bonds with amino acid residues Asn34, Arg126, and Trp412. [1]

Glucose uptake assay in cancer cells and in human red blood cells[1]
The inhibitory activity of compounds on glucose transport was analyzed by measuring the cell uptake of 2-deoxy-d-[3H] glucose as previously described.
Similar procedure was used for glucose uptake assay in human red blood cells (RBC), except that RBCs were washed and collected by centrifugation at 2,000 × g for 5 minutes as they are suspension cells, and the treated RBCs were solubilized in 0.1% SDS before radioactivity was measured.

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Cell proliferation (MTT) and clonogenic assays[1]
Cell proliferation and viability rates were measured using the MTT Proliferation Assay Kit (Cayman) or clonogenic assays.

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Hypoxia studies[1]
Cancer cell study in hypoxia was conducted using the Anaerobe Gas Generating Pouch System with indicator. The pouch formed an oxygen-free environment in which the compound-treated cells were incubated for 24 hours. After the hypoxic incubation, the treated cells were measured for their viability by the MTT assay.

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Glucose uptake assay in cancer cells: Cells were treated with WZB117 or vehicle, and then the uptake of 2-deoxy-D-[³H] glucose was measured. Cells were washed, lysed, and radioactivity was counted to determine glucose transport inhibition. [1]
Glucose uptake assay in human red blood cells (RBCs) and RBC-derived vesicles: RBCs were washed and collected by centrifugation. For vesicles (IOV and ROV), centrifugation at a higher speed was used to precipitate vesicles after washing. Treated RBCs or vesicles were solubilized before radioactivity measurement. [1]
Cell proliferation/viability assay: Cell proliferation and viability were measured using an MTT assay kit. Cells were seeded in plates, treated with WZB117, and after incubation, MTT reagent was added. The formed formazan crystals were solubilized, and absorbance was measured. [1]
Clonogenic assay: Cells were treated with WZB117, then a limited number of cells were re-seeded into culture dishes and allowed to grow into colonies for a period (e.g., 10-14 days). Colonies were fixed, stained, and counted to assess irreversible growth inhibition. [1]
Hypoxia study: Cells were treated with WZB117 and immediately transferred to an anaerobic pouch system to create an oxygen-free environment. Cells were incubated under hypoxia for 24 hours, and viability was then measured by MTT assay. [1]
Western blot analysis: Cells were lysed after treatment with WZB117. Lysates were subjected to SDS-PAGE, transferred to membranes, and probed with specific primary antibodies (e.g., against Glut1, phospho-Akt, phospho-mTOR, phospho-AMPK, GRP78/BiP, phospho-eIF2α, CHOP, PARP, CDK2, cyclin E2, phospho-Rb, hexokinase II, PKM2, PGAM1). After incubation with secondary antibodies, signals were detected. β-actin or β-tubulin was used as a loading control. [1]
Real-time PCR for Glut1 mRNA: Total RNA was isolated from treated A549 cells, reverse transcribed into cDNA, and then quantitative real-time PCR was performed using specific primer sets for human SLC2A1 (Glut1) and β-actin (as internal control). The δCt method was used for quantification. [1]
Lactate measurement: Extracellular lactate concentration in the culture media of treated cells was measured using a commercial lactate assay kit. [1]
Intracellular ATP measurement: Intracellular ATP levels in treated cells were measured using a luminescence-based ATP detection assay system. [1]
ATP rescue study: Various concentrations of ATP were added to the culture medium of cancer cells in the presence or absence of WZB117. After 24 hours, intracellular ATP levels and cell viability (by MTT assay) were measured. [1]
Cell-cycle analysis: Treated cells were fixed, stained with propidium iodide, and analyzed by flow cytometry to determine the distribution of cells in different cell-cycle phases (G0-G1, S, G2-M). [1]
Apoptosis/Necrosis detection by flow cytometry: Treated cells were stained with Annexin V-FITC and propidium iodide according to the manufacturer's instructions and then analyzed by flow cytometry to distinguish apoptotic (Annexin V-positive) and necrotic (propidium iodide-positive, Annexin V-negative) cells. [1]
Senescence-associated β-galactosidase (SA-β-gal) assay: Treated cells were fixed and stained using a commercial senescence β-galactosidase assay kit. Cells were observed under a microscope for the development of blue color (indicating β-gal activity) and enlarged morphology, which are markers of cellular senescence. [1]

Male NU/J nude mice of 6 to 8 weeks of age were purchased from The Jackson Laboratory and were fed with the Irradiated Teklad Global 19% protein rodent diet from Harlan Laboratories. To determine the in vivo anticancer efficacy of compound WZB117 on human tumor xenograft growth, NSCLC A549 cells in exponential growth phase were harvested, washed, precipitated, and resuspended in PBS. Each mouse was injected subcutaneously with 5 × 106 cancer cells in the flank. Compound treatment started 3 days after the cancer cells injection and when all tumors became palpable. Tumor cell–injected mice were randomly divided into 2 groups: control group (n = 10) treated with PBS/DMSO (1:1, v/v) and WZB117 treatment group (n = 10) treated with WZB117 (10 mg/kg body weight) dissolved in PBS/DMSO solution (1:1, v/v). Mice were given intraperitoneal injection with either PBS/DMSO vehicle or compound WZB117 (10 mg/kg) daily for 10 weeks. Tumor sizes were measured every 7 days with calipers, and tumor volume (L × W2/2) was calculated and presented as means ± SEM. All of the procedures involved in animal study were conducted in conformation with the guidelines of both Ohio University and NIH.[1]

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To evaluate in vivo anticancer efficacy, male NU/j nude mice (6-8 weeks old) were subcutaneously injected with 5 x 10⁶ human NSCLC A549 cells in the flank. Treatment started 3 days after cell inoculation when tumors were palpable. Mice were randomly divided into two groups: a control group (n=10) receiving daily intraperitoneal injections of PBS/DMSO (1:1, v/v) vehicle, and a treatment group (n=10) receiving daily intraperitoneal injections of WZB117 at 10 mg/kg body weight, dissolved in PBS/DMSO solution (1:1, v/v). Injections were administered daily for 10 weeks. Tumor sizes were measured weekly with calipers, and tumor volume was calculated. [1]

single intraperitoneal injection of WZB117 caused mild, transient hyperglycemia in mice, which returned to normal within 1 to 2 hours after injection, without causing persistent hyperglycemia. [1]
This publication does not provide other ADME/PK parameters (e.g., absorption, distribution, metabolism, excretion, half-life, bioavailability). [1]

In a 10-week animal study, mice treated with WZB117 (10 mg/kg/day, intraperitoneal injection) lost approximately 1 to 2 grams of body weight compared to mice treated with the carrier, with most of the weight loss attributed to a reduction in adipose tissue. [1] Blood cell counts at the end of the study showed changes in lymphocyte and platelet counts in mice treated with the compound compared to the control group, but all counts were within the normal range. [1]

[1]. A small-molecule inhibitor of glucose transporter 1 downregulates glycolysis, induces cell-cycle arrest, and inhibits cancer cell growth in vitro and in vivo. Mol Cancer Ther. 2012 Aug;11(8):1672-82.

WZB-117 is a diester formed by the condensation of two hydroxyl groups of 3-fluorocatechol with the carboxyl group of 3-hydroxybenzoic acid. It is a glucose transporter 1 (GLUT1) inhibitor that inhibits tumor growth in a mouse xenograft model. It has dual effects as an antitumor, a glucose transporter 1 inhibitor, and a radiosensitizer. WZB-117 is a diester belonging to the phenol, benzoate and monofluorobenzene class of compounds. Its function is related to 3-hydroxybenzoic acid and 3-fluorocatechol.

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WZB117 is a structural and functional analogue of a previously discovered class of glucose transporter inhibitors with higher activity. [1] The proposed anticancer mechanism involves immediate inhibition of Glut1-mediated glucose transport, leading to reduced glycolysis (lower ATP and lactate levels), thereby activating energy-sensing pathways (e.g., AMPK) and downregulating cell growth signaling pathways (Akt, mTOR). This ultimately leads to cell cycle arrest (mediated by decreased levels of cyclin E2 and pRb), followed by senescence and necrosis. Decreased intracellular ATP levels play a crucial role in the early inhibitory effect. [1] WZB117 showed synergistic effects with standard chemotherapy drugs and was superior to its non-tumorigenic counterparts under hypoxic conditions and against cancer cells. [1] This study suggests that WZB117 could serve as a prototype for further development of anticancer therapies targeting Glut1-mediated glucose transport and metabolism. [1]

C20H13FO6

368.32

368.069

C, 65.22; H, 3.56; F, 5.16; O, 26.06

1223397-11-2

46830365

Typically exists as white to off-white solids at room temperature

1.4±0.1 g/cm3

628.4±55.0 °C at 760 mmHg

333.9±31.5 °C

0.0±1.9 mmHg at 25°C

1.645

4.67

2

7

6

27

527

0

FC1C=CC=C(C=1OC(C1C=CC=C(C=1)O)=O)OC(C1C=CC=C(C=1)O)=O

FRSWCCBXIHFKKY-UHFFFAOYSA-N

InChI=1S/C20H13FO6/c21-16-8-3-9-17(26-19(24)12-4-1-6-14(22)10-12)18(16)27-20(25)13-5-2-7-15(23)11-13/h1-11,22-23H

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.79 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.79 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.79 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)