Ixazomib (MLN2238) , a second generation, boron-containing peptide proteasome inhibitor (PI), inhibits the chymotrypsin-like proteolytic (β5) site of the 20S proteasome (IC50/Ki: 3.4/0.93 nM, in cell-free assays), also inhibits the caspase-like (β1) and trypsin-like (β2) proteolytic sites (IC50: 31/3500 nM).

Caspase-like β1:31nM, Trypsin like β2:3500 nM, 20S proteasome:3.4 nM, 20S proteasome:0.93 nM(Ki)

MLN2238 is a biologically active metabolite of MLN9708.MLN2238 and Bortezomib are both time-dependent reversible inhibitors of the proteasome, but the isolated half-life of the proteasomal effect of MLN2238 (18 min) is 6-fold faster compared with that of Bortezomib (110 min).The oncological efficacy of MLN2238 is higher than that of Bortezomib. MLN2238 is a nitrogen-terminally capped dipeptide leucine borate that inhibits the hydrolysis site of chymotrypsin-like enzymes in the 20S proteasome (IC50/Ki: 3.4/0.93 nM). At higher concentrations, MLN2238 also inhibited the caspase-like hydrolysis site (IC50: 31 nM) and the trypsin-like hydrolysis site (IC50: 3.5 μM) of the 20S proteasome.MLN2238 inhibited Calu-6 cells (IC50: 9.7 nM).

MLN2238 is a biologically active metabolite of MLN9708.MLN2238 and Bortezomib are both time-dependent reversible inhibitors of the proteasome, but the isolated half-life of the proteasomal effect of MLN2238 (18 min) is 6-fold faster compared with that of Bortezomib (110 min).The oncological efficacy of MLN2238 is higher than that of Bortezomib. MLN2238 is a nitrogen-terminally capped dipeptide leucine borate that inhibits the hydrolysis site of chymotrypsin-like enzymes in the 20S proteasome (IC50/Ki: 3.4/0.93 nM). At higher concentrations, MLN2238 also inhibited the caspase-like hydrolysis site (IC50: 31 nM) and the trypsin-like hydrolysis site (IC50: 3.5 μM) of the 20S proteasome.MLN2238 inhibited Calu-6 cells (IC50: 9.7 nM).

Kinase assay : Calu-6 cells are cultured in MEM containing 10% fetal bovine serum and 1% penicillin/streptomycin and plated 1 day before the start of the experiment at 1 × 104 cells per well in a 384-well plate. Proteasome activity is assessed by monitoring hydrolysis of the chymotrypsin-like substrate Suc-LLVY-aminoluciferin in the presence of luciferase using the Proteasome-Glo assay reagents according to the manufacturer's instructions. Luminescence is measured using a LEADseeker instrument.

Calu-6 cells are cultured in MEM containing 10% FBS and 1% penicillin/streptomycin and plated 1 day before the start of the experiment at 1 × 104 cells per well in a 384-well plate. For IC50 determinations, cells are treated with varying concentrations of Bortezomib or MLN2238 in DMSO (0.5% final, v/v) for 1 hour at 37 °C. For reversibility experiments, cells are treated with either 1 μM Bortezomib or MLN2238 for 30 minutes at 37 °C and then washed thrice in medium to remove the Bortezomib or MLN2238. Cells are incubated for an additional 4 hours at 37 °C, after which the medium is removed and replaced with fresh medium. (Only for Reference)