GLS1 (IC50 = 23 nM); GLS1 (IC50 = 28 nM); GLS2 (IC50 >1 μM)

Telaglenastat (CB-839) (0.1-1000 nM; 72 hours) exhibits antiproliferative activity with IC50s of 49 nM and 26 nM, respectively, in MDA-MB-231 and HCC1806 cells[1].
Telaglenastat (CB-839) (1 μM; 72 hours) auses MDA-MB-231 and HCC1806 cells to undergo apoptosis by activating caspase 3/7[1].

Telaglenastat (CB-839) (200 mg/kg; p.o.; twice daily for 28 days) exhibits antitumor activity in xenograft models of TNBC[1].

The assay buffer, which contains 50 mM Tris-Acetate pH 8.6, 150 mM K2HPO4, 0.25 mM EDTA, 0.1 mg/mL bovine serum albumin, 1 mM DTT, 2 mM NADP+, and 0.01% Triton X-100, is used to measure the enzymatic activity. In order to quantify inhibition, glutamine and glutamate dehydrogenase (GDH) are first pre-mixed with the inhibitor (prepared in DMSO), and reactions are then started by adding rHu-GAC. 2 nM rHu-GAC, 10 mM glutamine, 6 units/mL GDH, and 2% DMSO are present in the final reactions. On a SpectraMax M5e plate reader, NADPH generation is tracked every minute for 15 minutes using fluorescence (Ex340/Em460 nm). Using a standard NADPH curve, relative fluorescence units (RFU) are converted to units of NADPH concentration (µM). Every assay plate has control reactions that track how GDH converts glutamate (1–75 µM) + NADP+ to α-ketoglutarate + NADPH. GDH stoichiometrically converts up to 75 µM glutamate to α-ketoglutarate/NADPH under these assay conditions. Fitting a straight line to the first five minutes of each progress curve yields the initial reaction velocities. A four-parameter dose response equation of the following form is used to fit inhibition curves: % activity = Bottom + (Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)).

In order to perform viability assays, all cell lines are exposed to CB-839 for 72 hours at the indicated concentrations. Cell Titer Glo is then used to measure any antiproliferative effects.

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Western blotting[3]
The samples were homogenized in 0.1% SDS buffer containing 10 mM EDTA, 125 mM NaCl, 25 mM HEPES, 0.5% deoxycholic acid, 10 mM Na3VO4, 0.1% SDS, 1% Triton X-100 with Complete™ protease inhibitor cocktail. The cell lysate was centrifuged at 12,000 rpm for 15 min. Then the supernatant-contained protein was collected and the protein concentration was tested by protein assay kit. The collected protein was separated on SDS-PAGE gel, and transferred onto PVDF membrane. The membrane was blocked with 5% skim milk for 1 h to reduce non-specific binding. Then, the membrane was incubated with one of the following rabbit polyclonal primary antibodies: anti-LC3B-I&II, anti-Beclin-1, anti-p62, anti-β-actin, anti-Tublin, anti-C-myc, anti-N-myc, anti-L-myc, anti-GLS and anti-ERα at 4 °C for 12 h. After 3 times of washes, the blot was incubated with secondary antibody HRP-conjugated goat anti-rabbit IgG for 1 h at room temperature. Finally, the signal was detected by the enhanced chemiluminescence kit and exposed to X-film.

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Quantitative real-time polymerase chain reaction (qRT-PCR)[3]
The cells were collected to extract total RNA by Trizol and then 500 ng of RNA was reverse-transcribed in accordance with the specification of FastKing RT Kit. According to the gene sequences, Primer 5.0 was used to design the primers, which were produced by Shanghai Sangon Biological Engineering Technology & Services Company. The reaction conditions of qRT-PCR were as follows: operating at 95 °C for 15 min once and then 40 cycles under 95 °C for 30 s, 60 °C for 45 s, 72 °C for 1 min. The reaction system was as follows (25 μl): 12.5 μl of Premix Ex Taq or SYBR Green Mix, 1 μl of forward primer, 1 μl of reverse primer, 1–4 μl of DNA template and ddH2O. The relative quantification (RQ) of target genes was calculated by using the following formula: RQ = 2-ΔΔCt, and the result was used for statistical analysis.

Female nu/nu mice with age 4–6 weeks (TNBC patient-derived xenograft model)[1]
200 mg/kg
Oral administration; twice daily for 28 days

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All animal experiments were approved by the Animal Ethics Committee of Shanghai General Hospital and were implemented in accordance with the Guide for the Care and Use of Laboratory Animals. Pathogen-free four-week-old female nude mice were obtained from Slaccas Animal Laboratory. The steps were as follows: Construct Xenograft model by subcutaneous injection of Ishikawa cells (2 × 106 in phosphate-buffered saline containing 50% Matrigel, n = 6 for each group). Implant estrogen pellets (60-d time release, 0.72-mg β-estradiol/pellet; subcutaneously unless otherwise noted. Formulate CB-839 solution with a concentration of 20 mg/mL in vehicle. The vehicle consists 25% hydroxypropyl-β-cyclodextrin (HPBCD) in 10 mmol/L citrate; and pH is 2. The dose volume for all groups is 10 mL/kg. When the volume of tumors reaches approximately 100–150 mm3, dose the mice orally twice a day (every 12 h) with the vehicle or the 200 mg/kg CB-839 prepared in vehicle. Take records of the volume of tumors every 3 days after transplantation: tumor volume = length×width2 / 2. Record the tumor weight and profile after sacrificing.[3]

[1]. Antitumor activity of the glutaminase inhibitor CB-839 in triple-negative breast cancer. Mol Cancer Ther. 2014 Apr;13(4):890-901.

[2]. Compensatory metabolic networks in pancreatic cancers upon perturbation of glutaminemetabolism. Nat Commun. 2017 Jul 3;8:15965.

[3]. Estrogen inhibits autophagy and promotes growth of endometrial cancer by promoting glutamine metabolism. Cell Commun Signal. 2019 Aug 20;17(1):99.

Telaglenastat is under investigation in clinical trial NCT02071862 (Study of the Glutaminase Inhibitor CB-839 in Solid Tumors).
Telaglenastat is an orally bioavailable inhibitor of glutaminase, with potential antineoplastic activity. Upon oral administration, CB-839 selectively and irreversibly inhibits glutaminase, a mitochondrial enzyme that is essential for the conversion of the amino acid glutamine into glutamate. By blocking glutamine utilization, proliferation in rapidly growing cells is impaired. Glutamine-dependent tumors rely on the conversion of exogenous glutamine into glutamate and glutamate metabolites to both provide energy and generate building blocks for the production of macromolecules, which are needed for cellular growth and survival.

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Glutamine serves as an important source of energy and building blocks for many tumor cells. The first step in glutamine utilization is its conversion to glutamate by the mitochondrial enzyme glutaminase. CB-839 is a potent, selective, and orally bioavailable inhibitor of both splice variants of glutaminase (KGA and GAC). CB-839 had antiproliferative activity in a triple-negative breast cancer (TNBC) cell line, HCC-1806, that was associated with a marked decrease in glutamine consumption, glutamate production, oxygen consumption, and the steady-state levels of glutathione and several tricarboxylic acid cycle intermediates. In contrast, no antiproliferative activity was observed in an estrogen receptor-positive cell line, T47D, and only modest effects on glutamine consumption and downstream metabolites were observed. Across a panel of breast cancer cell lines, GAC protein expression and glutaminase activity were elevated in the majority of TNBC cell lines relative to receptor positive cells. Furthermore, the TNBC subtype displayed the greatest sensitivity to CB-839 treatment and this sensitivity was correlated with (i) dependence on extracellular glutamine for growth, (ii) intracellular glutamate and glutamine levels, and (iii) GAC (but not KGA) expression, a potential biomarker for sensitivity. CB-839 displayed significant antitumor activity in two xenograft models: as a single agent in a patient-derived TNBC model and in a basal like HER2(+) cell line model, JIMT-1, both as a single agent and in combination with paclitaxel. Together, these data provide a strong rationale for the clinical investigation of CB-839 as a targeted therapeutic in patients with TNBC and other glutamine-dependent tumors.[1]

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Pancreatic ductal adenocarcinoma is a notoriously difficult-to-treat cancer and patients are in need of novel therapies. We have shown previously that these tumours have altered metabolic requirements, making them highly reliant on a number of adaptations including a non-canonical glutamine (Gln) metabolic pathway and that inhibition of downstream components of Gln metabolism leads to a decrease in tumour growth. Here we test whether recently developed inhibitors of glutaminase (GLS), which mediates an early step in Gln metabolism, represent a viable therapeutic strategy. We show that despite marked early effects on in vitro proliferation caused by GLS inhibition, pancreatic cancer cells have adaptive metabolic networks that sustain proliferation in vitro and in vivo. We use an integrated metabolomic and proteomic platform to understand this adaptive response and thereby design rational combinatorial approaches. We demonstrate that pancreatic cancer metabolism is adaptive and that targeting Gln metabolism in combination with these adaptive responses may yield clinical benefits for patients.[2]

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Background: Excessive estrogen exposure is an important pathogenic factor in uterine endometrial cancer (UEC). Recent studies have reported the metabolic properties can influence the progression of UEC. However, the underlying mechanisms have not been fully elucidated. Methods: Glutaminase (GLS), MYC and autophagy levels were detected. The biological functions of estrogen-MYC-GLS in UEC cells (UECC) were investigated both in vivo and in vitro. Results: Our study showed that estrogen remarkably increased GLS level through up-regulating c-Myc, and enhanced glutamine (Gln) metabolism in estrogen-sensitive UEC cell (UECC), whereas fulvestrant (an ER inhibitor antagonist) could reverse these effects. Estrogen remarkably promoted cell viability and inhibited autophagy of estrogen sensitive UECC. However, CB-839, a potent selective oral bioavailable inhibitor of both splice variants of GLS, negatively regulated Gln metabolism, and inhibited the effects of Gln and estrogen on UECC's growth and autophagy in vitro and / or in vivo. Conclusions: CB-839 triggers autophagy and restricts growth of UEC by suppressing ER/Gln metabolism, which provides new insights into the potential value of CB-839 in clinical treatment of estrogen-related UEC.[3]

C26H24F3N7O3S

571.57

571.161

C, 54.63; H, 4.23; F, 9.97; N, 17.15; O, 8.40; S, 5.61

1439399-58-2

Telaglenastat hydrochloride;1874231-60-3

71577426

Off-white to yellow solid powder

1.430±0.06 g/cm3

1.635

2.61

2

12

12

40

812

0

S1C(N([H])C(C([H])([H])C2=C([H])C([H])=C([H])C([H])=N2)=O)=NN=C1C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1C([H])=C([H])C(=NN=1)N([H])C(C([H])([H])C1C([H])=C([H])C([H])=C(C=1[H])OC(F)(F)F)=O

PRAAPINBUWJLGA-UHFFFAOYSA-N

InChI=1S/C26H24F3N7O3S/c27-26(28,29)39-20-9-5-6-17(14-20)15-22(37)31-21-12-11-18(33-34-21)7-1-2-10-24-35-36-25(40-24)32-23(38)16-19-8-3-4-13-30-19/h3-6,8-9,11-14H,1-2,7,10,15-16H2,(H,31,34,37)(H,32,36,38)

N-[6-[4-[5-[(2-pyridin-2-ylacetyl)amino]-1,3,4-thiadiazol-2-yl]butyl]pyridazin-3-yl]-2-[3-(trifluoromethoxy)phenyl]acetamide

Telaglenastat; CB839; Telaglenastat; 1439399-58-2; 2-(pyridin-2-yl)-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide; Telaglenastat [USAN]; U6CL98GLP4; CB839; N-[6-(4-{5-[2-(pyridin-2-yl)acetamido]-1,3,4-thiadiazol-2-yl}butyl)pyridazin-3-yl]-2-[3-(trifluoromethoxy)phenyl]acetamide; CB-839; CB 839

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: 10 mg/mL (17.50 mM) in 20% HP-β-CD/10 mM citrate pH 2.0 (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: 4 mg/mL (7.00 mM) in 70% PEG300 30% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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: 5 mg/mL (8.75 mM) in 20% SBE-β-CD/10 mM Trisodium citrate adjusted to pH 2.0 with HCL (add these co-solvents sequentially from left to right, and one by one), clear solution; Need ultrasonic and adjust pH to 2 with 1M HCl and heat to 55°C.

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Solubility in Formulation 4: 5% DMSO +Corn oil : 3mg/mL

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