Cell experiment:

Cells are incubated in serum-free RPMI media for 1-1.5 hours. Isolated human B cells are incubated with Spebrutinib at a final concentration of 0.001, 0.01, 0.1 and 1 μM. Ramos cells are incubated with 0.1 nM-3 μM Spebrutinib. Cells are then incubated in the presence of compound for 1 hour at 37℃. Following incubation, cells are centrifuged and resuspended in 100 μL of serum-free RPMI and BCR is stimulated with addition of 5 μg/mL α-human IgM. Samples are centrifuged, washed in phosphate-buffered saline (PBS), and lysed in 100 μL of Cell Extraction Buffer plus 1:10 (v/v) PhosSTOP Phosphatase Inhibitor and 1:10 (v/v) Complete Protease Inhibitor. Antibodies used for immunoblot analysis include P-PLCγ2, PLCγ2 (3871; CST), Syk (2712; CST), P-Syk (2710; CST), Btk, P-Btk, and Tubulin. Membranes are scanned on a Li-Cor Odyssey scanner using infrared fluorescence detection[1].

Spebrutinib besylate (AVL-292 benzenesulfonate; CC-292 besylate) is a potent inhibitor of Btk kinase activity (IC50<0.5 nM, Kinact/Ki=7.69×104 M-1s-1s) in biochemical assays. IC50:<0.5 nM (Btk)[1]

Spebrutinib (CC-292) is a covalent, highly selective, orally active inhibitor of Btk with IC50 value of 0.5 nM. Spebrutinib also less potently inhibits Yes, c-Src, Brk, Lyn, and Fyn with IC50s of 723 nM, 1.729 μM, 2.43 μM, 4.4 μM, and 7.15 μM, rspectively. Extensive analysis has revealed that the EC50 of Btk occupancy from a Spebrutinib dose-response in Ramos cells (EC50=6 nM) correlated directly with the cellular EC50 of Btk kinase inhibition with Spebrutinib (EC50=8 nM). Furthermore, the concentration at which Spebrutinib inhibits 90% of Btk activity in Ramos cells is 35 nM while the concentration of Spebrutinib required for 90% occupancy of Btk is 39 nM[1].

[1]. Evans EK, et al. Inhibition of Btk with CC-292 provides early pharmacodynamic assessment of activity in mice and humans. J Pharmacol Exp Ther. 2013 Aug;346(2):219-28.