Torin 2 is an mTOR inhibitor with EC₅₀ of 0.25 nM for inhibiting cellular mTOR activity, and exhibits 800-fold selectivity over PI3K (EC₅₀: 200 nM). Torin 2 also inhibits DNA-PK with an IC₅₀ of 0.5 nM in the cell free assay. Torin 2 can suppress both mTORC1 and mTORC2.

Torin 2 is subject to further profiling against a panel of lipid kinases with IC₅₀s of 2.81 nM, 0.5 nM, 5.67 nM, 8.58 nM, 18.3 nM, 24.5 nM and 28.1 nM for mTOR, DNA-pK, p110γ, hVPS34, PI4Kβ, PI3K-C2β and PI3K-C2α, respectively. Torin 2 (Torin2) possesses a 250 pM EC₅₀ for inhibiting mTOR in cells while maintaining 800-fold cellular selectivity relative to inhibition of PI3K and most other protein kinases^[1]. Torin 2 (Torin2) exhibits potent biochemical and cellular activity against PIKK family kinases including ATM (EC₅₀ 28 nM), ATR (EC₅₀ 35 nM) and DNA-PK (EC₅₀ 118 nM). Torin 2 potently inhibits T308 of Akt, a direct substrate of PDK1 and an indirect substrate of PI3Ks, with an EC₅₀ of less than 10 nM^[2]. Torin-2 can suppress both mTORC1 and mTORC2^[4].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Torin 2 (Torin2) exhibits good bioavailability and exposure and can maintain strong inhibition of mTOR activity in lung and liver to at least six hours after a single dose of 20 mg/kg. Torin 2 is easier to produce on scale and exhibits improved pharmacokinetic properties which should enable it use in vivo experiments^[1]. Torin 2 (Torin2) strongly suppresses pS6K(T389) and p4EBP1(T37/46) and partly suppresses pAkt(T308). Treatment of mice with AZD6244 at 25 mg/kg results in a profound inhibition of pERK. Combined administration of Torin 2 (40 mg/kg) and AZD6244 (25 mg/kg) demonstrates strong inhibition of all pharmacodynamics markers^[2]. Treatment with Torin 2 (Torin2) and Rapamycin induces IL-6 secretion by astrocytes and may contribute to the reduction of mechanical hypersensitivity after SCI. Torin1 and Torin 2 treatment increases IL-6 mRNA, suggesting that the PI3K-mTOR pathway is a negative regulator of IL-6 expression in astrocytes. Importantly, Torin 2 treatment does not show any cell toxicity, as no signs of cell death are observed by TUNEL assay or by detection of cleaved-caspase 3 by western blotting^[3].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

432.40

C₂₄H₁₅F₃N₄O

1223001-51-1

Room temperature in continental US; may vary elsewhere.

DMSO : 15.62 mg/mL (36.12 mM; Need ultrasonic)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% 1-Methyl-2-pyrrolidinone    90% PEG300

  Solubility: ≥ 2 mg/mL (4.63 mM); Clear solution
• 2.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 1.56 mg/mL (3.61 mM); Clear solution

  此方案可获得 ≥ 1.56 mg/mL (3.61 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 15.6 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• [1]. Liu Q, et al. Discovery of 9-(6-aminopyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (Torin2) as a potent, selective, and orally available mammalian target of rapamycin (mTOR) inhibitor for treatment of cancer. J Med Chem. 2011 Mar 10;54(5):1473-80.

  [2]. Liu Q, et al. Characterization of Torin2, an ATP-competitive inhibitor of mTOR, ATM, and ATR. Cancer Res. 2013 Apr 15;73(8):2574-86.

  [3]. Codeluppi S, et al. Interleukin-6 secretion by astrocytes is dynamically regulated by PI3K-mTOR-calcium signaling. PLoS One. 2014 Mar 25;9(3):e92649.

  [4]. Wang X, et al. mTORC signaling in hematopoiesis. Int J Hematol. 2016 May;103(5):510-8.

HCT116 cells are treated with 100 nM Torin 2 or AZD8055 for 1 hour before they are thoroughly washed out by 3×PBS and 1×DMEM medium. Then cells are incubated in DMEM medium for indicated time before they are lysed and collected using M-PER. Protein concentrations are measured and equal amount of proteins are loaded. Experiments are repeated three times and one set of results^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
Six-week old male C57BL/6 mice are fasted overnight prior to Torin 2 treatment. The mice are treated with vehicle (for 10 h) or Torin 2 (20 mg/kg for 6h) via oral gavage and then re-fed 1 h prior to sacrifice (CO₂ asphyxiation). Liver and lung are collected and frozen on dry ice. The frozen tissue is thawed on ice and lysed by sonication in tissue lysis buffer (50 mM HEPES, pH 7.4, 40 mM NaCl, 2 mM EDTA, 1.5 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM sodium β-glycerophosphate, 0.1% SDS, 1.0% sodium deoxycholate, and 1.0% Triton, supplemented with protease inhibitor cocktail tablets). The concentration of clear lysate is measured using the Bradford assay and samples are subsequently normalized by protein content and analyzed by SDS-PAGE and immunoblotting.
Rats^[3]
Female rats (220 g) are group-housed 4 animals per cage, and kept on a 12-hour light/dark cycle with food and water ad libitum. Spinal cord injury is done with the Keck Center for Neurosciences impactor using a 10 g weight dropped from a height of 25 mm onto the dorsal surface of the exposed spinal cord. After recording BBB scores, withdrawal thresholds evoked by touch stimulus, and body weights for the first week post-injury, animals are divided into 5 treatment groups: naïve (N=4), sham (N=6), vehicle (N=6), Torin 2 (N=6), and Torin 2+Rapamycin (N=8). Torin 2 alone (4 mg/kg) or in combination with Rapamycin (1.5 mg/kg) is administered orally by gavage once a day starting at day 15 after injury and ending at day 29. In sham operated rats only the laminectomy is performed.

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Liu Q, et al. Discovery of 9-(6-aminopyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (Torin2) as a potent, selective, and orally available mammalian target of rapamycin (mTOR) inhibitor for treatment of cancer. J Med Chem. 2011 Mar 10;54(5):1473-80.

  [2]. Liu Q, et al. Characterization of Torin2, an ATP-competitive inhibitor of mTOR, ATM, and ATR. Cancer Res. 2013 Apr 15;73(8):2574-86.

  [3]. Codeluppi S, et al. Interleukin-6 secretion by astrocytes is dynamically regulated by PI3K-mTOR-calcium signaling. PLoS One. 2014 Mar 25;9(3):e92649.

  [4]. Wang X, et al. mTORC signaling in hematopoiesis. Int J Hematol. 2016 May;103(5):510-8.