Ibrutinib

2 years -80 in solvent

Administered via p.o.

Chronic lymphocytic leukemia (CLL) cells

0.01-100 uM

PCI-32765 is dissolved in DMSO.

PCI-32765 shows the potent and irreversible inhibitory effect and selectivity for Btk enzymatic activity. In BCR pathway-activated DOHH2 cell line, PCI-32765 inhibits autophosphorylation of Btk, phosphorylation of Btk's physiological substrate PLCgamma, and phosphorylation of further downstream kinase, ERK with IC50 of 11 nM, 29 nM and 13 nM, respectively. [1], PCI-32765 exhibits a significant dose-dependent and time-dependent induction of cytotoxicity in chronic lymphocytic leukemia (CLL) cells. In addition, PCI-32765 induces cell death depending on caspase pathway activation and antagonizes the ability of CLL cells to proliferate after TLR signaling. [2], A recent study shows that PCI-32765 inhibits BCR-activated primary B cell proliferation with IC50 of 8 nM and results in inhibition of TNFalpha, IL-1beta and IL-6 production in primary monocytes with IC50 of 2.6 nM, 0.5 nM and 3.9 nM, respectively. [3]

48 hours

MTT (3', [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) assays are performed to determine cytotoxicity. Briefly, cells (CLL B cells or healthy volunteer T cells or NK cells) are incubated for 48 hours with different concentrations of PCI-32765, or vehicle control. MTT reagent is then added, and plates are incubated for an additional 20 hours before washing with protamine sulfate in phosphate-buffered saline. Dimethyl sulfoxide is added, and absorbance is measured by spectrophotometry at 540 nm in a Labsystems plate reader. Cell viability is also measured at various time points with the use of annexin/PI flow cytometry. Data are analyzed with Expo-ADC32 software package. Results are expressed as the percentage of total positive cells over untreated control. Experiments examining caspase-dependent apoptosis includes the addition of 100M Z-VAD.

Ibrutinib is a potent and highly selective Brutons tyrosine kinase (Btk) inhibitor with IC50 of 0.5 nM in cell-free assays, modestly potent to Bmx, CSK, FGR, BRK, HCK, less potent to EGFR, Yes, ErbB2, JAK3, etc. Ibrutinib is applicable as a Btk ligand in the synthesis of a series of PROTACs including P13I.