Bisindolylmaleimide I (GF109203X) is a potent and highly selective protein kinase C (PKC) inhibitor with a Ki of 14 nM.

PKC:14 nM

Bisindolylmaleimide I作为一种与ATP竞争的PKC抑制剂，能够阻止由激活PKC的刺激引起的血小板聚集，并且有作为研究PKC在信号传导途径中作用的工具的潜力。[1] Bisindolylmaleimide I对P-糖蛋白和MRP介导的多药耐药性具有逆转活性。[2] [3] Bisindolylmaleimide I通过抑制PKC显著减少了卡巴胆碱激活的ERK1/2的活化以及随后SNU-407结肠癌细胞的增殖。[4]

GF109203X（10 μg/只鼠，i.pl.）在Wistar大鼠中剂量依赖性地抑制BK引起的机械性痛觉过敏。[5]

Assay of protein kinase C: Protein kinase C is arrayed by measuring 32PI transferred from [gamma-32PI] ATP to lysine-rich histone type Ill-s. The reaction mixture (80 μL) contained 50 mM Tris-HCI. pH 7.4, 100 μM CaCl2, 10 mM MgCI2, 37.5 μL/mL histone type Ill-s, 10 μM [gamma-32PI] ATP (1250 cpm/pmol), 31 μM bovine brain phosphatidylserine and 0.5 μM 1,2 sn-dioleylglycerol. Fifteen μL of purified PKC (final concentration in assay 0.38 μg/mL) is added to the incubation mixture. After 10 min at 30°C, the reaction is stopped by addition of 30 μL of casein 30 mg/mL and 0.9 ml of 12% trichloroacetic acid. The acid precipitable material is collected by centrifugation, dissolved in 1N NaOH (100μL) and precipitated again with 1 ml of 12% trichloroacetic acid. The pellet is dissolved in 1N NaOH (100μL) and 32P incorporation is measured by scintillation counting in Aquasol.

Cell proliferation is monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells are seeded in 96-well plates and allowed to grow overnight. The cells are serum-starved for 18–24 hours and then treated with 1 mM carbachol for 48 hours in 100 μL serum-free RPMI 1640. Inhibitors are added 30 min prior to carbachol treatment. Following the treatment, 10 μL of MTT solution (5 mg/ml) is applied to each well, and the plates were incubated for 3 h at 37 °C. After the medium is removed, the formazan crystals formed are solubilized in 100 μL DMSO. The absorbance at 570 nm is measured using a microplate reader and the background absorbance at 690 nm is subtracted. Each assay is performed in triplicate. (Only for Reference)