CAL-101 inhibits PI3Kδ with high selectivity. (A) Chemical structure of CAL-101. (B) CAL-101 in vitro activity profiles (half-maximal inhibitory concentration values) against recombinant enzymes of class I, II, III, and IV PI3Ks. CAL-101 was diluted in dimethyl sulfoxide at a concentration of 10mM, and 10-point kinase inhibitory activities were measured over a concentration range (5.0-10⁴nM) with adenosine triphosphate at a concentration consistent with each enzymes K_ms. (C) Potency of CAL-101 in PI3K class I isoform-specific cell-based assays. For the analysis of p110α and p110β signaling, murine embryo fibroblasts were stimulated with PDGF or LPA and soluble protein was analyzed by Western blotting for Akt and pAkt⁴⁷³ levels. For the analysis of p110δ and p110γ signaling, basophil activation was measured in isolated peripheral blood mononuclear cell or whole blood using the Flow2 CAST kit according to the manufacturer's standardized methods (Buhlman Laboratories AG). p110δ was activated with anti-FCϵRI, and p110γ was activated with formyl-methionyl-leucyl-phenylalanine. To monitor the basophil cell population and cellular activation, anti–CD63-FITC and anti–CCR3-phycoerythrin antibodies were added to each sample. Cells were fixed and analyzed on a FC500MPL flow cytometer (Beckman Coulter).