STO-609

STO-609 inibisce le attività delle isoforme ricombinanti CaM-KKα e CaM-KKβ, con valori di Ki di 80 e 15 ng/ml, rispettivamente, e inibisce anche le loro attività di autofosforilazione. Questo composto è altamente selettivo per CaM-KK senza alcun effetto significativo sulle chinasi CaM a valle (CaM-KI e -IV). Nelle cellule HeLa trasfettate, questa sostanza chimica sopprime l'attivazione di CaM-KIV indotta da Ca²⁺ in modo dose-dipendente. Può permeare le cellule ed è un inibitore competitivo di ATP[1].

La somministrazione in vivo di STO-609 porta a un aumento degli osteoblasti e a una diminuzione degli osteoclasti, conferendo una protezione significativa dall'osteoporosi indotta dall'ovariectomia (OVX) nei topi adulti[2]. La somministrazione ICV di questo composto in vivo non ha influenzato le risposte controrregolatorie alla neuroglucopenia e all'attivazione di AMPK indotta dalla glucopenia[3].

• Linee cellulari

  HeLa cells

• Concentrazioni

  0.01-10 μg/ml

• Tempo di incubazione

  6 h

• Metodo

  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.

• Modelli animali

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

• Dosaggi

  10 μmol/kg

• Somministrazione

  i.p.