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

在多种实体瘤和血液移植瘤中,MLN9708有抗癌活性.

MLN9708与水溶液和血浆接触后, MLN9708快速水解为具有生物活性的MLN2238。MLN2238抑制20S蛋白酶体的糜蛋白酶类水解位点（IC50=3.4 nM,Ki=0.93 nM）。MLN2238也抑制20S蛋白酶体的caspase类水解的位点（IC50=31 nM）和胰蛋白酶类水解的位点（IC50=3.5 μM）。

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)