PI-1840（IC50 = 27 nM）is a reversible and selective chymotrypsin-like (CT-L) inhibitor, with little proteasome proteolytic effects on trypsin-like (T-L) and postglutamyl-peptide-hydrolysis-like (PGPH-L).

Chymotrypsin-like proteasome:27 nM

PI-1840在完整的人类MDA-MB-468癌细胞中强效抑制蛋白酶体CT-L活性，其IC50为0.37 μM，并抑制人类MDA-MB-468细胞的增殖/存活。[1] 在完整的癌细胞中，PI-1840抑制CT-L活性，诱导蛋白酶体底物p27、Bax和IκB-α的积累，抑制存活途径和活性，并诱导凋亡。[2]

PI-1840 (150 mg/kg i.p.) 在小鼠中抑制了MDA-MB-231乳腺肿瘤的生长。[2]

CT-L, T-L, PGPH-L proteolytic activity assays: In the high-throughput screen, fluorogenic peptides are used as substrates to assay (at 10 μM) the 50,000 compounds library from ChemBridge for inhibitory activity against the CT-L proteolytic activity of the purified rabbit 20S proteasome. To test for selectivity for CT-L over T-L and PGPH-L the corresponding fluorogenic peptides are used. Briefly, 1 nM of purified 20S rabbit proteasome is incubated with 20 μM Suc-Leu-Leu-Val-Tyr-AMC for the CT-L activity, Bz-Val-Gly-Arg-AMC for the T-L activity, and benzyloxycarbonyl Z-Leu-Leu-Glu-AMC for the PGPH-L activity for 1 h at 37 °C in 100 μL of assay buffer (50 mM Tris-HCl, pH 7.6) with or without compound. After incubation, production of hydrolyzed 7-amido-4-methyl-coumarin (AMC) groups is measured using a WALLAC Victor2 1420 Multilabel Counter with an excitation filter of 355 nm and an emission filter of 460 nm. The amount of AMC released is within the linear range. Bortezomib is used as a positive control for IC50 determinations. To determine proteasome activity in whole cell, extracts (5 μg) from cultured cell lysate is used instead of 20S rabbit proteasome, and followed the same assay mentioned above.

Cells are plated in 96-well plates in 100 μL medium and allowed to attach overnight. Cells are then incubated for 120 h with varying concentrations of inhibitors. Media is aspirated and replaced with 100 μL complete media containing 1 mg/ml MTT and incubated for three hours at 37 °C in 5% CO2 humidified incubator. Media is then aspirated and DMSO is added. Cells are incubated for 10 min at room temperature while shaking, and the absorbance is determined at 540 nm using a μQuant spectrophotometric plate reader. The IC50 values are determined using equation under CT-L, T-L, PGPH-L proteolytic activity assays.(Only for Reference)