XL388 is a selective inhibitor of mTOR with IC50 value of 9.9 nM [1].
mTOR, a member of the phosphatidylinositol 3-kinase (PI3K) cell survival pathway, plays an important role in the regulation of cell growth and proliferation by monitoring nutrient availability, cellular energy levels, oxygen levels, and mitogenic signals [1].
In MCF-7 cells, XL388 blocks mTORC1 phosphorylation of p70S6K with an IC50 value of 94 nM and mTORC2 phosphorylation of AKT with an IC50 value of 350 nM. When assayed alone, XL388 inhibits the viability of hematopoietic tumor cell lines in vitro. Also, XL388 synergizes with chemotherapeutics in cell-based assays to block cell viability [1].
XL388 shows robust antitumor activity in multiple xenograft models when dosed orally once daily in mice, especially in the MCF-7 xenograft model it shows > 100% tumor growth inhibition [1]. XL388 displays strong pharmacokinetics and good oral exposure in multiple species. Oral administration of XL388 to athymic nude mice implanted with MCF-7 xenograft tumors afforded significant and dose-dependent antitumor activity, which suggest that block of mTOR signaling is a viable target for antitumor activity [2].
参考文献:
[1]. Nicole Miller. Abstract B146: XL388: A novel, selective, orally bioavailable mTORC1 and mTORC2 inhibitor that demonstrates pharmacodynamic and antitumor activity in multiple human cancer xenograft models. Mol Cancer Ther, 2009, 8(12 Suppl):B146.
[2].Takeuchi CS, Kim BG, Blazey CM, et al. Discovery of a Novel Class of Highly Potent, Selective, ATP-Competitive, and Orally Bioavailable Inhibitors of the Mammalian Target of Rapamycin (mTOR). J Med Chem, 2013, 56(6):2218-34.
Kinase experiment: | The measurement of mTOR enzyme activity is performed in an ELISA format following the phosphorylation of 4E-BP1 protein. All experiments are performed in the 384-well format. Generally, 0.5 μL of DMSO containing varying concentrations of the test compound is mixed with 15 μL of the enzyme solution. Kinase reactions are initiated with the addition of 15 μL of a solution containing the substrate. The assay conditions are as follows: 0.2 nM mTOR, 10 μM ATP, and 50 nM NHis-tagged 4E-BP1 in 20 mM Hepes, pH 7.2, 1 mM DTT, 50 mM NaCl, 10 mM MnCl2, 0.02 mg/mL BSA, 0.01% CHAPS, 50 mM β-glycerophophate. Following an incubation of 120 min at ambient temperature, 20 μL of the reaction mixture is transferred to a Ni-chelate-coated 384-well plate. The binding step of the 4E-BP1 protein proceeded for 60 min, followed by washing four times each with 50 μL of Tris-buffered saline solution (TBS). Anti-phospho-4E-BP1 rabbit immunoglobulin G (IgG; 20 μL, 1:5000) in 5% BSA-TBST (0.2% Tween-20 in TBS) is added, and the reaction mixuture is further incubated for 60 min. Incubation with a secondary horseradish peroxidase (HRP)-tagged anti-IgG is similarly performed after the primary antibody is washed off (four washes of 50 μL). Following the final wash step with TBST, 20 μL of SuperSignal ELISA Femto is added and the luminescence measured using an EnVision plate reader. Data are reported as the mean (n≥2) [1]. |
Cell experiment: | U2OS, SaOs-2 and MG-63 OS cell lines as well as the murine calvaria-derived osteoblastic MC3T3-E1 cells are maintained and culture. The OB-6 human osteoblastic cells are cultured. For primary culture of murine osteoblasts, the trimmed calvariae of neonatal mice are digested with 0.1% collagenase I and 0.25% dispase. The resolving cell suspensions are neutralized with complete culture medium and are filtered. The calvarial osteoblasts are then resuspended in 10 mL α-MEM containing 15% FBS, and are cultured. Cells (5×104/well) are suspended in 1 mL of DMEM with 1% agar, 10 % FBS and with indicated XL388 (5, 25, 100 and 200nM) treatment. The cell suspension is then added on top of a pre-solidified 1% agar in a 100 mm culture dish. The drug containing medium is refreshed every 2 days. After 10-day incubation, the number of remaining colonies are stained and manually counted[2]. |
Animal experiment: | Mice, Rats, Dogs and Monkeys[1]Pharmacokinetic studies of XL388 are determined in female athymic nude mice, female CD rats, male beagle dogs, and male cynomolgus monkeys. XL388 is administered intravenously and by oral gavage at 10 mg/kg as a solution formulated in EPW (5% ethanol/45% PEG400/water+1:2 HCl (m/m)) to mice, 3 mg/kg as a solution formulated in EPW (5% ethanol/45% PEG400/water+1:2 HCl (m/m)) to CD rats and male beagle dogs, and 3 mg/kg as a solution formulated in EPW (5% ethanol/45% PEG400/water+1:1.5 HCl (m/m)) to male cynomolgus monkeys. The plasma levels of XL388 are monitored over a 24 h period. |
参考文献: [1]. Takeuchi CS, et al. Discovery of a novel class of highly potent, selective, ATP-competitive, and orally bioavailable inhibitors of the mammalian target of rapamycin (mTOR). J Med Chem. 2013 Mar 28;56(6):2218-34. | |
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