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

体外活性：CB-839 表现出时间依赖性且缓慢可逆的动力学。与 rHu-GAC 预孵育 1 小时后，CB-839 抑制谷氨酰胺酶的 IC50 值 < 50 nmol/L，比 BPTES 至少低 13 倍。 CB-839 在三阴性乳腺癌 (TNBC) 细胞系 HCC-1806 中具有抗增殖活性，而在雌激素受体阳性细胞系 T47D 中未观察到抗增殖活性。激酶测定：酶活性在含有 50 mM Tris-Acetate pH 8.6、150 mM K2HPO4、0.25 mM EDTA、0.1 mg/mL 牛血清白蛋白、1 mM DTT、2 mM NADP+ 和 0.01% Triton X-100 的测定缓冲液中测量。为了测量抑制作用，首先将抑制剂（在 DMSO 中制备）与谷氨酰胺和谷氨酸脱氢酶 (GDH) 预混合，并通过添加 rHu-GAC 引发反应。最终反应包含 2 nM rHu-GAC、10 mM 谷氨酰胺、6 单位/mL GDH 和 2% DMSO。在 SpectraMax M5e 读板仪上每分钟通过荧光 (Ex340/Em460 nm) 监测 NADPH 的生成，持续 15 分钟。使用 NADPH 标准曲线将相对荧光单位 (RFU) 转换为 NADPH 浓度单位 (μM)。每个检测板都包含对照反应，可监测 GDH 将谷氨酸（1 至 75 µM）加 NADP+ 转化为 α-酮戊二酸加 NADPH。在这些测定条件下，高达 75 µM 的谷氨酸通过 GDH 按化学计量转化为 α-酮戊二酸/NADPH。通过将每条进度曲线的前 5 分钟拟合成一条直线来计算初始反应速度。抑制曲线符合以下形式的四参数剂量反应方程：％活性=底部+（顶部-底部）/（1+10^（（LogIC50-X）* HillSlope））。细胞测定：对于活力测定，所有细胞系（HCC1806、MDA-MB-231 和 T47D 细胞）均用所示浓度的 CB-839 处理 72 小时，并使用 Cell Titer Glo 分析抗增殖作用。

在小鼠 TNBC 模型中，单剂 CB-839（200 mg/kg，口服）相对于载体对照可抑制肿瘤生长 61%。在小鼠 JIMT-1 异种移植模型中，单独使用 CB-839（200 mg/kg，口服）相对于载体对照、CB-839（200 mg/kg，口服）与紫杉醇（10 mg/kg，口服）很大程度上抑制了肿瘤的再生长，导致相对于媒介物对照的 TGI 为 100%。

测定缓冲液含有 50 mM Tris-Acetate pH 8.6、150 mM K2HPO4、0.25 mM EDTA、0.1 mg/mL 牛血清白蛋白、1 mM DTT、2 mM NADP+ 和 0.01% Triton X-100，用于测量酶活性。为了量化抑制作用，首先将谷氨酰胺和谷氨酸脱氢酶 (GDH) 与抑制剂（在 DMSO 中制备）预混合，然后通过添加 rHu-GAC 开始反应。最终反应中存在 2 nM rHu-GAC、10 mM 谷氨酰胺、6 单位/mL GDH 和 2% DMSO。在 SpectraMax M5e 读板机上，使用荧光 (Ex340/Em460 nm) 每分钟跟踪 NADPH 生成情况，持续 15 分钟。使用标准 NADPH 曲线，将相对荧光单位 (RFU) 转换为 NADPH 浓度单位 (μM)。每个检测板都有对照反应，可追踪 GDH 如何将谷氨酸 (1–75 µM) + NADP+ 转化为 α-酮戊二酸 + NADPH。在这些测定条件下，GDH 按化学计量将高达 75 µM 谷氨酸转化为 α-酮戊二酸/NADPH。将直线拟合到每条进度曲线的前五分钟即可得出初始反应速度。使用以下形式的四参数剂量反应方程来拟合抑制曲线：％活性=底部+（顶部-底部）/（1+10^（（LogIC50-X）* HillSlope））。

为了进行活力测定，所有细胞系均以指定浓度暴露于 CB-839 72 小时。然后使用 Cell Titer Glo 来测量任何抗增殖作用。

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蛋白质印迹[3]
将样品在含有10 mM EDTA，125 mM NaCl，25 mM HEPES，0.5%脱氧胆酸，10 mM Na3VO4、0.1%SDS、1%Triton X-100与Complete™蛋白酶抑制剂混合物。细胞裂解物在12000下离心 rpm持续15 分钟。然后收集含有蛋白质的上清液，并用蛋白质测定试剂盒检测蛋白质浓度。收集的蛋白质在SDS-PAGE凝胶上分离，并转移到PVDF膜上。膜用5%脱脂奶封闭1 h以减少非特异性结合。然后，将膜与以下兔多克隆一级抗体之一孵育：抗LC3B-I&II、抗Beclin-1、抗p62、抗β-肌动蛋白、抗Tublin、抗C-myc、抗N-myc、反L-myc、GLS和抗ERα °C持续12 h.洗涤3次后，将印迹与第二抗体HRP缀合的山羊抗兔IgG孵育1小时 h。最后，通过增强化学发光试剂盒检测信号，并将其暴露于X膜

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定量实时聚合酶链式反应（qRT-PCR）[3]
收集细胞以通过Trizol提取总RNA，然后500 ng的RNA根据FastKing RT试剂盒的说明书进行逆转录。根据基因序列，采用引物5.0设计引物，由上海三光生物工程技术服务公司生产。qRT-PCR的反应条件如下：在95 °C持续15 至少一次，然后40 95以下循环 °C持续30 s、 60 °C持续45 s、 72 °C 1 min。反应体系如下（25 μl）：12.5 μl预混料Ex Taq或SYBR绿色混合物，1 μl正向引物，1 μl反向引物，1-4 μl DNA模板和ddH2O。通过使用以下公式计算靶基因的相对定量（RQ）：RQ = 2-ΔΔCt，并将结果用于统计分析。

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

160.12

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)

配方 1 中的溶解度: 10 mg/mL (17.50 mM) in 20% HP-β-CD/10 mM citrate pH 2.0 (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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配方 2 中的溶解度: 4 mg/mL (7.00 mM) in 70% PEG300 30% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: 5 mg/mL (8.75 mM) in 20% SBE-β-CD/10 mM Trisodium citrate adjusted to pH 2.0 with HCL (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 需要超声波并用 1M HCl 将 pH 调节至 2，并加热至 55°C。

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配方 4 中的溶解度: 5% DMSO +Corn oil : 3mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。