AZD5363 (Capivasertib)

Capivasertib (AZD5363) is a potent Akt inhibitor with IC50 of 3 nM, 8 nM and 8 nM for Akt1, Akt2 and Akt3, respectively. [1] It inhibits phosphorylation of AKT substrates in cells with a potency of approximately 0.3 to 0.8 μM. This compound inhibits the proliferation of 41 of 182 solid and hematologic tumor cell lines with a potency of < 3 μM. [2] Activating mutations in PIK3CA, loss or inactivation of tumor suppressor PTEN, or HER2 amplification all are significantly predictive of responsiveness to AZD5363. Additionally, correlation is also seen between the RAS mutation status of cell lines and resistance to it. [1]

Oral dosing of Capivasertib (AZD5363) (100, 300 mg/kg) to nude mice causes dose- and time-dependent reduction of PRAS40, GSK3β, and S6 phosphorylation in BT474c xenografts, reversible increases in blood glucose concentrations, and dose-dependent decreases in 2[¹⁸F]fluoro-2-deoxy-d-glucose (¹⁸F-FDG) uptake in U87-MG xenografts. Chronic oral dosing of this compound (130, 200, and 300 mg/kg) causes dose-dependent growth inhibition of xenografts derived from various tumor types, including HER2+ breast cancer models.

• Caliper Off-Chip Incubation Mobility Shift-assay

  Het vermogen van Capivasertib (AZD5363) en andere verbindingen om de activiteit van AKT1, AKT2 en AKT3 te remmen, wordt geëvalueerd met de Caliper Off-Chip Incubation Mobility Shift-assay. Actieve recombinante AKT1, AKT2 of AKT3 worden geïncubeerd met een 5-FAM-gelabeld, op maat gesynthetiseerd peptidesubstraat samen met toenemende concentraties van deze verbinding. De uiteindelijke reacties bevatten 1 tot 3 nM AKT1-, AKT2- of AKT3-enzymen; 1,5 mM peptidesubstraat; ATP op K _m voor elke AKT-isoform; 10 mM MgCl₂, 4 mM DTT, 100 mM HEPES en 0,015% Brij-35. De reacties worden gedurende 1 uur bij kamertemperatuur geïncubeerd en gestopt door toevoeging van buffer die 100 mM HEPES, 0,015% Brij-35-oplossing, 0,1% coatingreagens, 40 mM EDTA en 5% DMSO bevat. Platen worden vervolgens geanalyseerd met een Caliper LC3000, waardoor scheiding van peptidesubstraat en gefosforyleerd product mogelijk is door elektroforese met daaropvolgende detectie en kwantificering van laser-geïnduceerde fluorescentie.

• Cellijnen

  182 solid and hematologic tumor cell lines

• Concentraties

  ~30 μM

• Incubatietijd

  72 hours

• Methode

  Cell proliferation assay is determined by 2 methods, MTS and Sytox Green, using Capivasertib (AZD5363). Briefly, cells are seeded in 96-well plates and incubated overnight at 37 ℃, 5% CO₂. Cells are then exposed to concentrations of this compound ranging from 30 to 0.003μM for 72 hours. For the MTS endpoint, cell proliferation is measured by the CellTiter AQueous Non-Radioactive Cell Proliferation Assay reagent in accordance with the manufacturer's protocol. For the Sytox Green endpoint, Sytox Green nucleic acid dye diluted in TBS-EDTA buffer is added to cells (final concentration of 0.13 μM) and the number of dead cells detected using an Acumen Explorer. Cells are then permeabilized by the addition of saponin (0.03% final concentration, diluted in TBS-EDTA buffer), incubated overnight and a total cell count measured. Predose measurements are made for both MTS and Sytox Green endpoints, and concentration needed to reduce the growth of treated cells to half that of untreated cells values are determined using absorbance readings (MTS) or live cell counts.

• Dierlijke modellen

  Female nude mice and male SCID mice with BT474c, U87MG, KPL-4, HCC-1187 xenografts.

• Doseringen

  130 mg/Kg - 300 mg/Kg

• Toediening

  p.o.