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.

• Med Sci Monit 26 (2020): e924393.
• MMed Sci Monitor (2020): e924393-1.

AEB071 is an inhibitor of protein kinase C (PKC). The PKC inhibitor which can block the T-cell activation has the ability of immune suppression [1].

The protein kinase C (PKC) isoforms is very important in cell signaling, proliferation, differentiation, migration, survival, and death. PKC family has many isoforms. Among the PKC isoforms, PKC isoforms have basal effect on the T cells’ activation and other immune cell functions [2,3].

ABE071 is a potent inhibitor of novel and classical PKC isoforms. Through the inhibition of PKC, AEB071 can depress the activation and proliferation of T-cell and decrease the production of cytokine.ABE071 can also suppress the NK cell activity. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression

AEB071 is an effective treatment strategy for the cure of autoimmune diseases. According to the Psoriasis Area Severity Index (PASI) score, after 2 weeks’ treatment with 300 mg bid AEB071, Clinical severity of psoriasis was reduced up to 69% compared with baseline[2,3].

References:
[1]. Weckbecker G1, Pally C, Beerli C, et al. Effects of the novel protein kinase C inhibitor AEB071 (Sotrastaurin) on rat cardiac allograft survival using single agent treatment or combination therapy with cyclosporine, everolimus or FTY720. Transpl Int. 2010 May 1;23(5):543-52
[2]. Skvara H1, Dawid M, Kleyn E, Wolff B, et al. The PKC inhibitor AEB071 may be a therapeutic option for psoriasis. J Clin Invest. 2008 Sep;118(9):3151-9.
[3]. Matz M1, Weber U, Mashreghi MF, et al. Effects of the new immunosuppressive agent AEB071 on human immune cells. Nephrol Dial Transplant. 2010 Jul;25(7):2159-67