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].

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].

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.

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.

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]

[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 resulting from the formal condensation of the two hydroxy groups of 3-fluorocatechol with the carboxy groups of 3-hydroxybenzoic acid. It is an inhibitor of glucose transporter 1 (GLUT1) that suppresses tumour growth in mouse xenograft models. It has a role as an antineoplastic agent, a glucose transporter 1 inhibitor and a radiosensitizing agent. It is a diester, a member of phenols, a benzoate ester and a member of monofluorobenzenes. It is functionally related to a 3-hydroxybenzoic acid and a 3-fluorocatechol.

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.)