Inhibition of mTOR is evaluated using two methodologies: The high-throughput assay uses an α screen capture complex technology with a recombinant truncated FLAG-tagged mTOR (amino acids 1362-2549; expressed in HEK293 cells) and a biotinylated p70 peptide substrate. In addition, native mTOR activity is assayed using immunoprecipitation of full-length mTOR from HeLa cytoplasmic extract, and the endogenous mTOR is in protein complexes with Rictor and Raptor. A kinase assay is performed in the presence of recombinant 4E-BP1 protein as substrate with detection of the phosphorylated product through an ELISA format. The activity of the lipid kinases, class I PI3Ks α, β, δ, and γ, is measured using recombinant PI3Ks with the lipid PIP2 as substrate. Assays for the ataxia-telangiectasia mutated (ATM) and DNA-PK are performed. Finally, counterscreen against 260 kinases is carried out at a fixed concentration of 10 μM AZD-8055^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

For growth inhibition and acridine staining, cells are exposed to increasing concentrations of AZD-8055 (0 to 1,280 nM) for 72 to 96 h and stained for cell nuclei (0.03 mg/mL Hoechst 33342) and acidic vesicles (1 μg/mL acridine orange). Images are captured at 450 and 536 nm on an ArrayScan II platform, and the percentage of acidic vesicles and the number of cells are quantified. For LC3 assessment, cells are exposed to e64d/pepstatin (10 μg/mL) for 30 to 90 min before incubation with AZD8055. Cells are lysed on ice and analyzed by immunoblotting^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
Tumor cells (10⁶ for U87-MG, 5×10⁶ for A549) are injected s.c. in a volume of 0.1 mL, and mice are randomized into control and treatment groups when tumor size reach 0.2 cm³. AZD-8055 is administered by oral gavage (0.1 mL/10 g of body weight) once or twice daily. The control group receive the vehicle only. Tumor volumes (measured by caliper), animal body weight, and tumor condition are recorded twice weekly for the duration of the study. The tumor volume is calculated.
Rats^[2]
Sprague-Dawley (SD) rats (250 g) and pregnant SD rats (16-18 days gestation, used for microglia extraction) are used. In experiment 1, 48 rats (54 rats are used, 48 rats survive after the surgery) are assigned randomly to four groups: sham group, SAH 3 h group, SAH 24 h group, SAH 72 h group. The animals in SAH 3 h, 24 h and 72 h groups are subjected to experimental SAH and are killed at 3 h, 24 h and 72 h after blood injection, respectively (n=12 for each group). In experiment 2, 72 rats (83 rats are used, 72 rats survive after the surgery) are assigned randomly to sham group (n=18), SAH+vehicle group (n=18), SAH+Rapamycin group (n=18), SAH+AZD8055 group (n=18). The rat receive a single intraperitoneal injection of Rapamycin immediately after induction of SAH, the dose of Rapamycin used is 150 μg/kg body weight. AZD8055 is administered by oral gavage with the dose of 14 mg/kg body weight. Rats in vehicle group are treated with equal volume solvent. All the rats in experiment 2 are killed at 24 h post-SAH. In experiment 3, enriched microglia are distributed into 5 groups: Control, OxyHb, OxyHb+vehicle (DMSO), OxyHb+Rapamycin and OxyHb+AZD8055. Twenty-four hours after microglia re-seeding, cells are treated with OxyHb (10 μM), DMSO (volume equal to Rapamycin and AZD8055), Rapamycin (2.74 mM), AZD8055 (0.8 nM) respectively in fresh medium. After incubation for 24 h (in 37°C, 5% CO₂), cell medium is removed, washed by PBS for 3 times and fixed by 4% paraformaldehyde.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Chresta CM, et al. AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity. Cancer Res, 2010, 70(1), 288-298.  [Content Brief]

  [2]. You W, et al. Inhibition of mammalian target of rapamycin attenuates early brain injury through modulating microglial polarization after experimental subarachnoid hemorrhage in rats. J Neurol Sci. 2016 Aug 15;367:224-31.  [Content Brief]

  [3]. Kawata T, et al. Dual inhibition of the mTORC1 and mTORC2 signaling pathways is a promising therapeutic target for adult T-cell leukemia. Cancer Sci. 2017 Oct 27.  [Content Brief]