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.

• Saggio di spostamento della mobilità per incubazione fuori chip Caliper

  La capacità di Capivasertib (AZD5363) e di altri composti di inibire l'attività di AKT1, AKT2 e AKT3 viene valutata mediante il saggio Caliper Off-Chip Incubation Mobility Shift. L'AKT1, AKT2 o AKT3 ricombinante attiva viene incubata con un substrato peptidico sintetizzato su misura marcato con 5-FAM insieme a concentrazioni crescenti di questo composto. Le reazioni finali contenevano da 1 a 3 nM di enzimi AKT1, AKT2 o AKT3; 1,5 mM di substrato peptidico; ATP a K _m per ogni isoforma di AKT; 10 mM di MgCl₂, 4 mM di DTT, 100 mM di HEPES e 0,015% di Brij-35. Le reazioni vengono incubate a temperatura ambiente per 1 ora e interrotte mediante l'aggiunta di un tampone contenente 100 mM di HEPES, 0,015% di soluzione di Brij-35, 0,1% di reagente di rivestimento, 40 mM di EDTA e 5% di DMSO. Le piastre vengono quindi analizzate utilizzando un Caliper LC3000, consentendo la separazione del substrato peptidico e del prodotto fosforilato mediante elettroforesi con successiva rilevazione e quantificazione della fluorescenza indotta da laser.

• Linee cellulari

  182 solid and hematologic tumor cell lines

• Concentrazioni

  ~30 μM

• Tempo di incubazione

  72 hours

• Metodo

  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.

• Modelli animali

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

• Dosaggi

  130 mg/Kg - 300 mg/Kg

• Somministrazione

  p.o.