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

METHODS: C57BL/6J BMM cells were treated with CA-074 methyl ester (50 μM) for 24, 48 and 72 hours, and the target protein expression was detected by Western Blot.
RESULTS: CA-074 methyl ester inhibited RANKL-stimulated c-FOS upregulation and subsequent NFATc1 self-amplification. [1]

METHODS: To study the neuroprotective effect of CA-074 methyl ester, CA-074 methyl ester (1 μg, 10 μg) was administered to the hippocampus induced by global cerebral I/R injury.
RESULTS: CA-074 methyl ester prevented necroptosis in hippocampal CA1 neurons induced by global cerebral I/R injury. CA-074 methyl ester pretreatment strongly inhibited lysosomal membrane disruption and cathepsin-B leakage. CA-074 methyl ester inhibited the overexpression and nuclear translocation of RIP3 and the reduction of NAD+ levels after I/R injury. Pretreatment with CA-074 methyl ester enhanced the upregulation of Hsp70. [2]
METHODS: To study the effect of CA-074 methyl ester on osteoclastogenesis, CA-074 methyl ester (10, 30 mg/kg) was injected into the skull of mice.
RESULTS: CA-074 methyl ester inhibited osteoclastogenesis in vivo. [1]
METHODS: To study the anti-inflammatory activity of CA-074 methyl ester, CVB+CA-074 methyl ester (4 mg/kg) was injected intramuscuarly into guinea pigs.
RESULTS: Inflammation scores were significantly lower in the CVB+None group than in the CVB+ NONE group. The number of CD8+T cells was reduced in the CVB+CA-074 methyl ester group compared with the sham-operated group. [3]

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)