STO-609

STO-609 remt de activiteiten van recombinante CaM-KKα- en CaM-KKβ-isoformen, met Ki-waarden van respectievelijk 80 en 15 ng/ml, en remt ook hun autofosforyleringsactiviteiten. Deze verbinding is zeer selectief voor CaM-KK zonder significant effect op de stroomafwaartse CaM-kinasen (CaM-KI en -IV). In getransfecteerde HeLa-cellen onderdrukt deze chemische stof de Ca²⁺-geïnduceerde activering van CaM-KIV op een dosisafhankelijke manier. Het kan cellen doordringen en is een competitieve remmer van ATP[1].

In vivo toediening van STO-609 resulteert in verhoogde osteoblasten en verminderde osteoclasten, wat aanzienlijke bescherming biedt tegen door ovariectomie (OVX) geïnduceerde osteoporose bij volwassen muizen[2]. ICV-toediening van deze verbinding in vivo beïnvloedde de tegenregulerende responsen op neuroglucopenie en AMPK-activering geïnduceerd door glucopenie niet[3].

• Cellijnen

  HeLa cells

• Concentraties

  0.01-10 μg/ml

• Incubatietijd

  6 h

• Methode

  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.

• Dierlijke modellen

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

• Doseringen

  10 μmol/kg

• Toediening

  i.p.