In vitro activity: Sotrastaurin (< 10μM) treatment effectively abrogated at low nanomolar concentration markers of early T-cell activation, such as interleukin-2 secretion and CD25 expression, in primary human and mouse T cells. Sotrastaurin (200 nM) inhibits the CD3/CD28 antibody- and alloantigen-induced T-cell proliferation responses in the absence of nonspecific antiproliferative effects. Sotrastaurin (<3 μM) markedly inhibits lymphocyte function-associated antigen-1-mediated T-cell adhesion. Sotrastaurin(< 20 μM) selectively impair the proliferation of CD79 mutant ABC DLBCL cell lines, correlating with decreased NF-κB signaling avctivity. AEB071 at concentration of 5 μM induces G1 arrest and/or cell death in CD79 mutant cells.

Kinase Assay: Classical and novel PKC isotypes are assayed by scintillation proximity assay technology. In brief, the assay is 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 is performed for 60 min at room temperature. The reaction is 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 is measured in a MicroBetaTrilux counter for 1 min. PKCζ is assayed. In situ Thr-219 autophosphorylation status analysis of PKCθ is done by a phospho-site-specific antibody.

Cell Assay: 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.

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