Torin 1 is an mTOR inhibitor that inhibits mTORC1 and mTORC2 (IC50=2/10 nM) selectively and ATP-competitively. Torin 1 induces cellular autophagy and possesses anti-tumor, anti-inflammatory, and anti-aging activities.

mTORC2:10 nM, mTORC1:2 nM

方法：野生型 MEFs 细胞用 Torin 1 (250 nm) 处理 4 天，使用 CellTiterGlo viability assay 检测细胞增殖。
结果：250 nm Torin1 完全抑制细胞增殖。[1]
方法：人类内分泌细胞系 BON 用 Torin 1 (62.5-150 µM) 处理 3-24 h，使用 Western Blot 检测靶点蛋白表达水平。
结果：Torin1 以剂量依赖的方式增加 NT 分泌；在 Torin1 处理后 3-24 h，NT 分泌增加。Torin1 处理降低了 Akt (S473) 磷酸化，p70S6K (T389) 以及 4E-BP1 (T37/46) 的磷酸化也受到抑制。[2]

方法：为检测体内抗肿瘤活性，将 Torin 1 (20 mg/kg，20% N-mentyl-2-pyrrolidone, 40% PEG400 and 40% water) 腹腔注射给携带人胶质母细胞瘤 U87MG 的 NCR nude 小鼠，每天一次，持续十天。
结果：连续给药十天导致大于 99% 的肿瘤生长抑制。停止药物治疗后，肿瘤继续生长，这表明 Torin 1 治疗主要是细胞抑制性的，并且大量肿瘤细胞在治疗过程中仍然存活。[3]

mTORC1 and mTORC2 in Vitro Kinase Assays: To produce soluble mTORC1, HEK-293T cell lines that stably express N-terminally FLAG-tagged Raptor are generated using vesicular stomatitis virus G-pseudotyped MSCV retrovirus. For mTORC2, similar HeLa cells that stably express N-terminally FLAG-tagged Protor-1 are generated. Both complexes are purified by lysing cells in 50 mm HEPES, pH 7.4, 10 mm sodium pyrophosphate, 10 mm sodium β-glycerophosphate, 100 mm NaCl, 2 mm EDTA, 0.3% CHAPS. Cells are lysed at 4 °C for 30 min, and the insoluble fraction is removed by microcentrifugation at 13,000 rpm for 10 min. Supernatants are incubated with FLAG-M2 monoclonal antibody-agarose for 1 h and then washed three times with lysis buffer and once with lysis buffer containing a final concentration of 0.5 M NaCl. Purified mTORC1 is eluted with 100 μg/mL 3×FLAG peptide in 50 mm HEPES, pH 7.4, 100 mm NaCl. Eluate can be aliquoted and stored at -80 °C. Substrates S6K1 and Akt1 are purified. Kinase assays are performed for 20 min at 30 °C in a final volume of 20 μL consisting of the kinase buffer (25 mm HEPES, pH 7.4, 50 mm KCl, 10 mm MgCl2, 500 μm ATP) and 150 ng of inactive S6K1 or Akt1 as substrates. Reactions are stopped by the addition of 80 μL of sample buffer and boiled for 5 min. Samples are subsequently analyzed by SDS-PAGE and immunoblotting.

Cell viability is assessed with the CellTiter-Glo Luminescent Cell Viability Assay. On Day 0, 96-well plates are seeded with 500 cells per well and grown overnight. On Day 1, cells are treated with the appropriate compounds and subsequently analyzed on Days 3-5. For analysis, plates are incubated for 60 min at room temperature; 50 μL of CellTiter-Glo reagent is added to each well, and plates are mixed on an orbital shaker for 12 min. Luminescence is quantified on a standard plate luminometer. (Only for Reference)