In vitro activity: Seletalisib is a potent, ATP-competitive, and selective PI3Kδ inhibitor able to block protein kinase B (AKT) phosphorylation following activation of the B-cell receptor in a B-cell line. Moreover, seletalisib inhibited N-formyl peptide-stimulated but not phorbol myristate acetate–stimulated superoxide release from human neutrophils, consistent with a PI3Kδ-specific activity. Findings from cellular assays of adaptive immunity demonstrated that seletalisib blocks human T-cell production of several cytokines from activated T-cells. Additionally, seletalisib inhibited B-cell proliferation and cytokine release. In human whole blood assays, seletalisib inhibited CD69 expression upon B-cell activation and anti-IgE-mediated basophil degranulation. From 239 kinases screened, seletalisib at a concentration of 10 μM showed no inhibitory activity greater than 47% (MAP4K4) against non-PI3K kinase enzymes. Against nonkinase enzymes, seletalisib showed weak activities against phosphodiesterase (PDE)3A, PDE2A1, and PDE4D2, with inhibition varying between 32 and 74% at 10 μM. When screened at a concentration of 10 μM against 55 receptors and ion channels, the highest inhibitory activity of seletalisib observed was 20%. One receptor, neuropeptide Y receptor (Y1) showed 54% activation. In vitro receptor binding and enzyme assays across a broad range of target classes showed that seletalisib is selective for PI3Kδ. Seletalisib potently inhibited the phosphorylation of AKT following anti-IgM stimulation of the BCR on Ramos cells with an IC50 of 15 nM. When profiled in a wide range of primary cell assay systems, including fibroblasts, epithelial, endothelial and vascular smooth muscle cells, seletalisib showed significant activity only in those systems containing lymphocytes, demonstrating its functional selectivity towards PI3Kδ-expressing cells.

Kinase Assay: Seletalisib is dissolved 1 mM solution in DMSO, and tested in a concentration response (seletalisib), to explore the effects of PI3Kδ-specific inhibition compared with complete inhibition of class I PI3K signaling. In addition, seletalisib is tested in the BioMap BT cell system at concentrations of 1000, 100, 10, and 1 nM. An activity profile is generated based on the effect of the compounds on the levels of cellular readouts, including cytokines, growth factors, adhesion molecules, and proliferation endpoints.

Cell Assay: Serially diluted seletalisib and goat anti-human F(ab)2 IgM in serum-free RPMI 1640 were added to Ramos cells plated in serum-free RPMI 1640. The plate was incubated for 10 minutes, chilled on ice, and the cells pelleted by centrifugation. Cellular phosphorylated AKT was detected by MSD assay.