AZD-8055

AZD-8055 is an orally bioavailable, highly selective, ATP-competitive mTOR kinase inhibitor that directly binds to the ATP-binding pocket of the mTOR kinase domain, while potently inhibiting both mTORC1 and mTORC2 complexes (IC₅₀ values of 0.8 nM and 0.1 nM, respectively). AZD-8055 is suitable for research in oncology, metabolic diseases, and fibrotic disorders.

mTOR:0.8 nM (MDA-MB-468 cells)

Methods: HPCAL1 was knocked down in Huh7 human hepatocellular carcinoma cells using shRNA. Following transfection, cells were treated with AZD-8055 at gradient concentrations (0, 10, 50, 100, 500 nM) for 72 hours. Cell viability was assessed using the CCK-8 assay.
Results: HPCAL1-knockdown cells exhibited increased sensitivity to AZD-8055 (lower cell viability).[1]
Methods: Uveal melanoma cell lines: 92.1, Mel202, MP38, MP41. AZD-8055 (1 μM) and romidepsin (25 ng/mL) were added. After 6 hours of romidepsin treatment, fresh medium was replaced, and cells were cultured for an additional 48 hours. Apoptosis was assessed by flow cytometry (Annexin V/SYTOX Green staining).
Results: The combination of AZD-8055 and romidepsin induced the highest level of apoptosis in all four cell lines, outperforming other combinations. [2]

Methods: A MYC/Trp53⁻/⁻ liver cancer model was established via hydrodynamic tail-vein injection. Hpcal1 was simultaneously knocked out using CRISPR/Cas9 (sgHpcal1) or a control (sgCON). Following successful model establishment, AZD-8055 (10 mg/kg/day, once daily) was administered via intraperitoneal injection for 30 consecutive days.
Results: AZD-8055 treatment significantly reduced tumor burden in sgHpcal1 mice, demonstrating superior efficacy compared to the sgCON group. [1]

The activity of mTOR was assayed using the recombinant mTOR technique described above. For inhibition experiments, AZD8055 was added to the reaction mixture and preincubated for 10 min before addition of ATP. Inhibition was performed at 1 to 3,000 nmol/L of AZD8055 in varying concentrations of ATP (40–200 μmol/L) [1].

For growth inhibition and acridine staining, cells were exposed to increasing concentrations of AZD8055 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 were captured at 450 and 536 nm on an ArrayScan II platform, and the percentage of acidic vesicles and the number of cells were quantified. For LC3 assessment, cells were exposed to e64d/pepstatin (10 μg/mL) for 30 to 90 min before incubation with AZD8055. Cells were lysed on ice and analyzed by immunoblotting [1].

Tumor cells (10^6 for U87-MG, 5 × 10^6 for A549) were injected s.c. in a volume of 0.1 mL, and mice were randomized into control and treatment groups when tumor size reached 0.2 cm^3. AZD8055 was formulated in 30% (w/v) captisol (pH 3.0). The control group received the vehicle only. Tumor volumes (measured by caliper), animal body weight, and tumor condition were recorded twice weekly for the duration of the study. The tumor volume was calculated (taking length to be the longest diameter across the tumor and width to be the corresponding perpendicular diameter) using the following formula: (length × width) × √(length × width) × (π/6) [1].

Ethanol: 3 mg/mL (6.44 mM), Sonication is recommended.

H2O: < 1 mg/mL (insoluble or slightly soluble)

DMSO: 91 mg/mL (195.47 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 4.5 mg/mL (9.67 mM), Solution.