STO-609

STO-609 inhibe les activités des isoformes recombinantes CaM-KKα et CaM-KKβ, avec des valeurs de Ki de 80 et 15 ng/ml, respectivement, et inhibe également leurs activités d'autophosphorylation. Ce composé est hautement sélectif pour CaM-KK sans effet significatif sur les CaM kinases en aval (CaM-KI et -IV). Dans les cellules HeLa transfectées, cette substance chimique supprime l'activation de CaM-KIV induite par le Ca²⁺ de manière dose-dépendante. Il peut perméer les cellules et est un inhibiteur compétitif de l'ATP[1].

L'administration in vivo de STO-609 entraîne une augmentation des ostéoblastes et une diminution des ostéoclastes, conférant une protection significative contre l'ostéoporose induite par l'ovariectomie (OVX) chez les souris adultes[2]. L'administration ICV de ce composé in vivo n'a pas affecté les réponses contre-régulatrices à la neuroglucopénie et l'activation de l'AMPK induite par la glucopénie[3].

• Lignées cellulaires

  HeLa cells

• Concentrations

  0.01-10 μg/ml

• Temps dincubation

  6 h

• Méthode

  HeLa cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were subcultured in 6-cm dishes 12 h before transfection. The cells were then transferred to serum-free medium and treated with a mixture of either 3 g of pME18s plasmid DNA or 3 g of HA(hemagglutinin-tagged)-CaM-KIV and 20 μg of LipofectAMINE reagent in 2.5 ml of medium. After 20 h of incubation, the cells were further cultured in serum-free medium for 6 h in either the absence or presence of various concentrations of STO-609 (0.01-10 μg/ml in Me₂SO at a final concentration of 0.5%) and then treated with or without 1 μM ionomycin for 5 min. Stimulation was terminated by the addition of 1 ml of lysis buffer, 2 mM EDTA, 2 mM EGTA, 1% Nonidet P-40, 10% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/liter leupeptin, 10 mg/liter trypsin inhibitor, and 1 μM microcystin LR), and the cells were lysed for 30min on ice. The cell extract was collected and centrifuged at 15,000×g for 15 min, the supernatant was precleared with 40 μl of Protein G-Sepharose for 2h at 4 °C, and the supernatant was mixed with 4 g of anti-HA antibody for 3h. 40 μl of Protein G-Sepharose was then applied to the extract and incubated overnight. The immunoprecipitated resin was washed three times with 1 ml of the lysis buffer and then washed with 1 ml of kinase buffer. Protein G-Sepharose with immunoprecipitated HA-CaM-KIV was subjected to the protein kinase assay in the presence of 1 mM EGTA using syntide-2 as a substrate.

• Modèles animaux

  WT, Camkk2−/− and Camk4−/− mice (all in C57BL/6 background)

• Posologies

  10 μmol/kg

• Administration

  i.p.