MLN2238 suppresses the chymotrypsin-like proteolytic (β5) site of the 20S proteasome(Ki50=0.93 nM, IC50=3.4 nM/). The biologically active form of MLN9708 (Ixazomib Citrate) is MLN2238 in aqueous solutions or plasma.

20S proteasome:0.93 nM(Ki), 20S proteasome:3.4 nM

MLN9708 exhibits anticancer activity in a variety of solid tumors and hematologic malignancies.

Upon contact with aqueous solutions and plasma, MLN9708 rapidly hydrolyzes into MLN2238, which possesses bioactivity. MLN2238 inhibits the chymotrypsin-like hydrolytic sites of the 20S proteasome ([IC50=3.4 nM,Ki=0.93 nM]). It also inhibits the caspase-like ([IC50=31 nM]) and trypsin-like hydrolytic sites ([IC50=3.5 μM]) of the 20S proteasome.

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)