Sotrastaurin (AEB071)

Binding assays

Classical and novel PKC isotypes were assayed by scintillation proximity assay technology. In brief, the assay was performed in 20 mM Tris-HCl buffer, pH 7.4, and 0.1% bovine serum albumin by incubating 1.5 μM of the peptide substrate with 10 μM [33P]ATP), 10 mM Mg (NO3)2, 0.2 mM CaCl2, and PKC at a protein concentration varying from 25 to 400 ng/ml, and lipid vesicles containing 30 mol% phosphatidylserine, 5 mol% diacylglycerol (DAG), and 65 mol% phosphatidylcholine at a final lipid concentration of 0.5 μM. Incubation was performed for 60 min at room temperature. The reaction was stopped by adding 50 μl of a mixture containing 100 mM EDTA, 200 μM ATP, 0.1% Triton X-100, and 0.375 μg/well streptavidin-coated scintillation proximity assay beads in PBS without Ca2+ and Mg2+. Incorporated radioactivity was measured in a MicroBetaTrilux counter for 1 min.

Cell lines

GNAQ/GNA11-mutant Uveal Melanoma cell lines

Preparation method

The solubility of this compound in DMSO is >21.9 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

125, 250, 500, 1000 nM; 5 days

Applications

AEB071 inhibited cell proliferation in GNAQ/GNA11-mutant Uveal Melanoma cell lines with inhibition of the PKC/ERK1/2 pathway.

Animal models

nu/nu SCID female mice bearing GNAQ mutant xenograft

Dosage form

Oral administration, 80mg/kg/d, three times daily

Application

AEB071/BYL719 combination inhibited in vivo tumor growth in a GNAQ mutant xenograft model.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Evenou J P, Wagner J, Zenke G, et al. The potent protein kinase C-selective inhibitor AEB071 (sotrastaurin) represents a new class of immunosuppressive agents affecting early T-cell activation[J]. Journal of Pharmacology and Experimental Therapeutics, 2009, 330(3): 792-801.

[2]. Musi E, Ambrosini G, De Stanchina E, et al. The phosphoinositide 3-kinase α selective inhibitor BYL719 enhances the effect of the protein kinase C inhibitor AEB071 in GNAQ/GNA11-mutant uveal melanoma cells[J]. Molecular cancer therapeutics, 2014, 13(5): 1044-1053.