Torkinib (PP 242) (PP 242) is a selective and ATP-competitive mTOR inhibitor (IC50: 8 nM). It also inhibits mTORC1/2 (IC50s: 30/58 nM).

mTOR:8 nM (cell free), mTORC1:30 nM (cell free), mTORC2:58 nM (cell free), p110δ:0.10 μM (cell free)

Torkinib（PP242）有效抑制了mTOR（IC50：8 nM），但对其他PI3-K家族成员的活性较低。通过对219种蛋白激酶的测试，显示出相对于蛋白质激酶组的显著选择性。在BT549细胞中，PP242抑制了Akt的磷酸化，mTOR底物p70S6K及其下游靶点S6[1]的磷酸化。PP242以低纳摩尔浓度显著抑制生长（>90%，GI50：12 nM）。与雷帕霉素相比，PP242在携带PI3K功能增益或PTEN功能丧失的固体肿瘤细胞系中表现出更强的抗增殖效力[2]。

在鼠p190模型中，短期口服Torkinib以剂量依赖的方式显著降低了脾脏和骨髓中的白血病负担。在一项长期生存研究中，口服Torkinib（30和60 mg/kg）显著推迟了白血病的发病[2]。在脂肪和肝脏中，Torkinib能完全抑制Akt在S473和T308的磷酸化。令人惊讶的是，在骨骼肌中，Torkinib只能部分抑制Akt的磷酸化，并且在抑制T308的磷酸化方面比S473更有效，尽管它能完全抑制4EBP1和S6的磷酸化[3]。

Purified kinase domains were incubated with inhibitors at 2- or 4-fold dilutions over a concentration range of 50 - 0.001 μM or with vehicle (0.1% DMSO) in the presence of 10 μM ATP, 2.5 μCi of γ-32P-ATP and substrate. Reactions were terminated by spotting onto nitrocellulose or phosphocellulose membranes, depending on the substrate; this membrane was then washed 5–6 times to remove unbound radioactivity and dried. Transferred radioactivity was quantitated by phosphorimaging and IC50 values were calculated by fitting the data to a sigmoidal doseresponse using Prism software [1].

Cells were seeded in triplicate wells of 96-well flat bottom culture plates for 48 hr in the presence of increasing concentrations of indicated inhibitors. Cell viability and median-effect dose affecting growth (GIC50) was determined using the MTS assay. Absorbance values (490 nm) were normalized to controls and expressed as %MTS conversion. Wells lacking cells but with MTS added was used as the zero value when normalizing. For drug combination experiments, a range of fixed ratios of inhibitors was used to assess synergy using the combination index (CI) with CalcuSyn software according to the median-effect method as previously described. For proliferation experiments with PC-3, SKOV3, 786-O, and U87 cells, the CellTiter-Glo Luminescent reagent was used following the manufacturer's instructions. Quantitation was performed as mentioned above [2].

Drugs were prepared in 100 μl of vehicle containing 20% DMSO, 40% PEG-400, and 40% saline. Six-wk-old male C57BL/6 mice were fasted overnight prior to drug treatment. PP242 (0.4 mg), rapamycin (0.1 mg), or vehicle alone was injected IP. After 30 min for the rapamycin-treated mouse or 10 min for the PP242 and vehicle-treated mice, 250 mU of insulin in 100 μl of saline was injected IP. 15 min after the insulin injection, the mice were killed by CO2 asphyxiation followed by cervical dislocation. Tissues were harvested and frozen on liquid nitrogen in 200 μl of cap lysis buffer. The frozen tissue was thawed on ice, manually disrupted with a mortar and pestle, and then further processed with a micro tissue-homogenizer. The protein concentration of the cleared lysate was measured by Bradford assay and 5–10 μg of protein was analyzed by Western blot as described above [3].