CA-074 methyl ester (Cathepsin B Inhibitor IV) is a selective inhibitor of Cathepsin B (IC50=36.3 nM). CA-074 methyl ester has neuroprotective, anti-inflammatory and anti-cancer effects.

Cathepsin B (pH 5.5):13.7 μM, Cathepsin B (pH 4.6):8.9 μM, Cathepsin B (pH 7.2):7.6 μM

After seven days of cell culture and osteoclast generation, the media is removed and washed three times with PBS. BMMs are fixed with a fixing solution supplied by the manufacturer. The cells are incubated at 37°C with a solution containing deionized water, Fast Garnet GBC, Napthol phosphate, Acetate, and Tartrate for 1 h. The staining solution is removed, washed with PBS (3×), and air-dried. TRAP positive cells with three or more nuclei across whole culture area are counted as multinucleated osteoclasts using light microscopy.

HL-60 cells are pre-treated for 24 h with 200 μM BSO followed by 60 min with 1 mM DEM. Thereafter, the cells(1×106/ml) are incubated with 100 μM CA-074 or CA-074Me, in the presence of 1% DMSO, 200 μM BSO and 1 mM DEM at 37°C. Control cultures are treated with 1% DMSO alone or with 100 μM Z-FA-DMK in the presence of 200 μM BSO and 1 mM DEM. Untreated HL-60 cells are incubated with 100 μM CA-074 or CA-074Me, in the presence of 1% DMSO at 37°C. Untreated control cultures are incubated with 1% DMSO alone or with 100 μM Z-FA-DMK. After 2 h incubation, cells are washed three times with PBS/1% glucose and lysed in 100 mM citrate, pH 5.0, 2% Chaps (106 cells/100 μl). Subsequently, the lysate is centrifuged and the clarified supernatant used to assay for proteolytic activity.(Only for Reference)