Cathepsin B; neuroprotective, anti-cancer, and anti-inflamatory

RANKL-induced osteoclastogenesis in BMM cells from C57BL/6J and NOD/ShiLtJ mice is inhibited by CA-074Me (5 μM and 50 μM). The anti-osteoclast activity of CA-074Me is observed 24 hours post-RANKL stimulation in vitro. It is noteworthy that this impact is not mediated by the MAPK-ERK signaling cascade. Both sodium-induced NFATc1 autoenhancement and RANKL-stimulated c-FOS upregulation are inhibited by CA-074Me [2]. CVB1-induced cell engraftment is inhibited by CA-074Me [3].

Before and after treatment, hippocampal CA1 neurons are protected against whole-brain I/R injury-induced programmed toluene by CA074-me (1 μg, 10 μg). CA074-me significantly inhibited cathepsin-B leakage and lysosomal membrane rupture. In addition to inhibiting the overexpression and nuclear translocation of RIP3, CA074-me also increased the upregulation of Hsp70 and decreased NAD+ levels following I/R injury [1]. When compared to the CVB+None group, the guinea pig group that received CA-074Me (4 mg/kg/day im) had a significantly higher central score. 30 mg/kg) prevents bone deterioration and osteoclastogenesis[2]. In the CVB+CA-074Me group, there were fewer CD8+ T cells than in the sham operation group [3].

Oxygesn–glucose deprivation (OGD) and DND-153 staining[1]
OGD was performed in glucose-free deoxygenated buffer medium inside an OGD chamber with a 95% N2 and 5% CO2 atmosphere at 37 °C for 2 h. The sham group was placed in a similar buffer containing 25 mM glucose and kept for 2 h in incubator. The CA074-me group was incubated in medium containing 1 μg CA074-me 1 h before OGD exposure. The change of lysosomal pH in hippocampal neurons 22 h after OGD injury was assessed by the staining of LysoSensor Green DND-153 . The cells were incubated in medium containing 1 μM LysoSensor Green DND-153 for 60 min followed by washing several times with the medium. Then the tissues were observed under an inverted fluorescence microscope or a confocal microscope.

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Tartrate Resistant Acid Phosphatase (TRAP) Assay[2]
The TRAP assay was performed using an Acid Phosphatase staining kit according to the manufacturer's instructions. After seven days of cell culture and osteoclast generation, the media was removed and washed three times with PBS. BMMs were fixed with a fixing solution supplied by the manufacturer. The cells were incubated at 37°C with a solution containing deionized water, Fast Garnet GBC, Napthol phosphate, Acetate, and Tartrate for 1 h. The staining solution was removed, washed with PBS (3×), and air-dried. TRAP positive cells with three or more nuclei across whole culture area were counted as multinucleated osteoclasts using light microscopy[2].
TRAP assay staining on calvaria histology sections was performed in a similar manner. The slides were washed with PBS (3×) and incubated at 37°C for 1 h in a solution containing deionized water, Fast Garnet GBC, Napthol phosphate, Acetate, and Tartrate from TRAP assay kit. The slides were washed with PBS (3×), air dried, and mounted using an aqueous mounting media. TRAP positive cells with three or more nuclei in the calvarial suture region were counted as multinucleated osteoclasts using light microscopy

Immunoblotting Analysis[2]
Cells were seeded on 6 well plates at a density of 0.2 × 106 cells/well, the culture conditions were the same as Section “Macrophage Isolation From Mouse Bone Marrow, Culture, and Osteoclast Formation”. The cells were stimulated with RANKL and/or CA074-me and washed twice with cold PBS. The cells were harvested with RIPA lysis buffer and sonicated. 20 μg of protein lysate was separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in Tris-Buffered Saline with Tween-20 (TBST) (25mM Tris/HCl, pH7.6, 150 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature and then incubated overnight with primary antibodies including NFATc1,, pERK1/2, ERK1/2, c-FOS, and GAPDH (Cell Signaling).42 The membrane was then washed with TBST (3× for 10 min) and incubated with Horseradish Peroxidase (HRP)-conjugated secondary antibody for 30 min. Proteins were visualized using a commercial HRP-detection reagent.

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Immunocytochemistry[2]
BMMs were cultured in 8-chamber slide with MEMα media supplemented with 30 ng/ml M-CSF for three days. Afterwards, BMMs were stimulated with 30 ng/ml M-CSF, 50ng/ml RANKL, with or without 50 μM CA074-me for 15 min. Cells were fixed in phosphate-buffered 4% paraformaldehyde, permeablized with 0.1% triton -100, and then stained with NF-κB p65 primary antibody (Abcam). Alexa Fluor 488 rabbit anti-mouse IgG was used as the secondary antibody.

In Vivo RANKL-Induced Osteoclastogenesis[2]
All animal procedures were performed in accordance with approved IACUC protocols. RANKL (0.08 mg/kg) with and without CA074-me (10 mg/kg or 30 mg/kg) were mixed in sterile, nonimmunogenic 1% Extracel-HP gel according to the manufacturer's instructions. The gel is composed of thiol-modified sodium hyaluronate, thiol-modified heparin, thiol-modified gelatin, and degassed deionized sterile water. The hydrogel mixture was prepared in an aseptic hood using a sterile syringe. The control sham hydrogel contained sterile Phosphate Buffered Saline (PBS) without any cytokines. The osteolysis group was given 0.08 mg/kg RANKL in a hydrogel to induce pathologic bone loss, as described in previously published papers.6, 16 The hydrogel-only, hydrogel-RANKL, and hydrogel-RANKL-CA-074Me mixture was injected into 8-week old male mice calvarium in an aseptic hood (n = 5) following general anesthesia (80 mg/kg of ketamine and 7 mg/kg of xylazine). After four days, the calvaria were excised, fixed in 4% formaldehyde for 24 h, decalcified in 20% EDTA for one week, and sectioned into slides from paraffin blocks. The slides underwent Tartrate-Resistant Acid Phosphatase (TRAP) staining (see section Tartrate Resistant Acid Phosphatase (TRAP) Assay) to identify osteoclasts.

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lobal cerebral I/R injury model and drug administration[1]
Four-vessel occlusion (4-VO) for global cerebral ischemia with minor modification as described in our previous study was used (Wang et al., 2011). Under 4% (w/v) choral hydrate (400 mg/kg) anesthesia, both vertebral arteries were permanently electro-cauterized and the bilateral common carotid arteries (CCAs) were freed from surrounding tissues. After closing the surgical incisions, rats were allowed to recover for 24 h. On the following day, anesthesia was induced with 4% isoflurane and the CCAs were occluded with aneurysm clips for 20-min to induce global cerebral ischemia, then the clips were removed for reperfusion. Rectal temperature was maintained at 37 ± 0.5 °C throughout the procedures. Rats were moved to the animal’s incubator to keep the proper temperature until fully awake. Rats with dilated pupils and without seizures were selected for experiments.[2]
CA074-me was dissolved in 0.9% NaCl containing 2% DMSO to 0.2 μg/μl or 2 μg/μl. 1 h before ischemia or 1 h post reperfusion, CA074-me (0.2 μg/μl or 2 μg/μl) or vehicle (2% DMSO) was injected into the right cerebral ventricle (anteroposterior −0.92; mediolateral 1.5; dorsoventral 3.5 mm) with a total volume of 5 μl at 0.5 μl/min. Rats were assigned to 5 groups: the I/R group received 5 μl vehicle; three CA074-me groups were subjected to the same procedures as the I/R group, and received 1 μg CA074-me (CA074-me 1 μg) or 10 μg CA074-me (CA074-me 10 μg) 1 h before 20-min ischemia and 1 μg CCA074-me 1 h post reperfusion (Post CA074-me 1 μg) respectively; the sham group was subjected to the same procedures as the I/R group, except for occlusion of the CCAs.

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Thirty-two female shorthair guinea pigs (body weight: 154±18 g and age: 4 weeks old) were used. The animal models of PM were established by injection with CVB1 as described previously.16 In brief, four experimental groups(each group contains eight guinea pigs) were included: (A) sham group: received normal saline; (B) CVB1+ None group: received CVB1+Freunds complete adjuvant (FCA); (C) CVB1+ Saline group (pseudo-intervention group): received CVB1+FCA+normal saline (NS); and (D) CVB1+CA074-me group: CVB1+FCA+CA-074Me. CA074-me (4 mg/kg/day i.m.) was given 24 h after CVB1 injection for 7 consecutive days. Four weeks after CVB1 injection, the animals were killed, and lung tissues were collected for the following experiments.[3]

[1]. Protective mechanisms of CA074-me (other than cathepsin-B inhibition) against programmed necrosis induced by global cerebral ischemia/reperfusion injury in rats. Brain Res Bull. 2016 Jan;120:97-105.

[2]. CA-074Me compound inhibits osteoclastogenesis via suppression of the NFATc1 and c-FOS signaling pathways. J Orthop Res. 2015 Oct;33(10):1474-86.

[3]. Treatment with CA-074Me, a Cathepsin B inhibitor, reduces lung interstitial inflammation and fibrosis in a rat model of polymyositis. Lab Invest. 2015 Jan;95(1):65-77.

CA-074 methyl ester (CA-074Me) is a cell-permeable cathepsin B inhibitor. It is converted by cellular esterases to CA-074.

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Many studies have demonstrated the key role of lysosomes in ischemic cell death in the brain and have led to the "lysosomocentric" hypothesis. In this hypothesis, the release of cathepsin-B due to a change of lysosomal membrane permeabilization (LMP) or rupture is critical, and this can be prevented by its inhibitors CA074 and CA074-me. However, the role of CA074-me in neuronal death and its effect on the change of lysosomal membrane integrity after global cerebral ischemia/reperfusion (I/R) injury is not clear, so we investigated this here. Rat hippocampal CA1 neuronal death was evaluated after 20-min global cerebral I/R injury. CA074-me (1 μg, 10 μg) were given intracerebroventricularly 1h before ischemia or 1h post reperfusion. The changes of heat shock protein 70 (Hsp70), cathepsin-B, lysosomal-associated membrane protein 1 (LAMP-1), receptor-interacting protein 3 (RIP3), and the change of lysosomal pH were evaluated respectively. Hippocampal CA1 neuronal programmed necrosis induced by global cerebral I/R injury was prevented by CA074-me both pre-treatment and post-treatment. Diffuse cytoplasmic cathepsin-B and LAMP-1 immunostaining synchronized with the pyknotic nuclear changes 2 days post reperfusion, and a rise of lysosomal pH with the leakage of DND-153, a dye of lysosomes, after oxygen-glucose deprivation (OGD) was detected. Both of these changes demonstrated the rupture of lysosomal membrane and the leakage of cathepsin-B, and this was strongly inhibited by CA074-me pre-treatment. The overexpression and nuclear translocation of RIP3 and the reduction of NAD(+) level after I/R injury were also inhibited, while the upregulation of Hsp70 was strengthened by CA074-me pre-treatment. Delayed fulminant leakage of cathepsin-B due to lysosomal rupture is a critical harmful factor in neuronal programmed necrosis induced by 20-min global I/R injury. In addition to being an inhibitor of cathepsin-B, CA074-me may have an indirect neuroprotective effect by maintaining lysosomal membrane integrity and protecting against lysosomal rupture.[1]

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The osteoclast is an integral cell of bone resorption. Since osteolytic disorders hinge on the function and dysfunction of the osteoclast, understanding osteoclast biology is fundamental to designing new therapies that curb osteolytic disorders. The identification and study of lysosomal proteases, such as cathepsins, have shed light on mechanisms of bone resorption. For example, Cathepsin K has already been identified as a collagen degradation protease produced by mature osteoclasts with high activity in the acidic osteoclast resorption pits. Delving into the mechanisms of cathepsins and other osteoclast related compounds provides new targets to explore in osteoclast biology. Through our anti-osteoclastogenic compound screening experiments we encountered a modified version of the Cathepsin B inhibitor CA-074: the cell membrane-permeable CA-074Me (L-3-trans-(Propylcarbamoyl) oxirane-2-carbonyl]-L-isoleucyl-L-proline Methyl Ester). Here we confirm that CA-074Me inhibits osteoclastogenesis in vivo and in vitro in a dose-dependent manner. However, Cathepsin B knockout mice exhibited unaltered osteoclastogenesis, suggesting a more complicated mechanism of action than Cathepsin B inhibition. We found that CA-074Me exerts its osteoclastogenic effect within 24 h of osteoclastogenesis stimulation by suppression of c-FOS and NFATc1 pathways.[2]

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Cathepsin B (CB) is involved in the turnover of proteins and has various roles in maintaining the normal metabolism of cells. In our recent study, CB is increased in the muscles of polymyositis/dermatomyositis (PM/DM). However, the role of CB in interstitial lung disease (ILD) has not been reported. ILD is a frequent complication of PM/DM, which is the leading cause of death in PM/DM. It carries high morbidity and mortality in connective tissue diseases, characterized by an overproduction of inflammatory cytokines and induced fibrosis, resulting in respiratory failure. The etiology and pathogenesis of ILD remain incompletely understood. This study investigated whether treatment with CA-074Me, a specific inhibitor of CB, attenuates ILD in PM. CB expression, inflammation, and fibrosis were analyzed in the lung tissues from patients with PM/DM. The animal model of PM was induced in guinea pigs with Coxsackie virus B1 (CVB1). CA-074Me was given 24 h after CVB1 injection for 7 consecutive days. At the end of the experiment, the animals were killed and lung tissues were collected for the following analysis. Inflammation, fibrosis and apoptosis cells, and cytokines were assessed by histological examinations and immunohistochemical analyses, western blot analysis and transferase-mediated dUTP nick-end labeling assay. In patients with PM/DM, the protein levels of CB were significantly elevated in lung tissues compared with healthy controls, which correlated with increases in inflammation and fibrosis. Similarly, the expression of CB, inflammation and fibrosis, CD8(+) T cell, CD68(+) cell, tumor necrosis factor-alpha, transforming growth factor-beta1 infiltrations, and apoptotic cell death were significantly increased in lung tissues of the guinea-pig model of CVB1-induced PM. These changes were attenuated by the administration of CA-074Me. In conclusion, this study demonstrates that PM/DM increases CB expression in lung tissues and inhibition of CB reduces ILD in a guinea-pig model of CVB1-induced PM. This finding suggests that CB may be a potential therapeutic target for ILD.[3]

C19H30N2O6

382.45

397.221

C, 59.67; H, 7.91; N, 7.32; O, 25.10

147859-80-1

134448-10-5

6610318

White to off-white solid powder

0.694

2

6

10

28

611

5

O1[C@@]([H])(C(N([H])C([H])([H])C([H])([H])C([H])([H])[H])=O)[C@@]1([H])C(N([H])[C@]([H])(C(N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(=O)OC([H])([H])[H])=O)[C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])=O

XGWSRLSPWIEMLQ-YTFOTSKYSA-N

InChI=1S/C19H31N3O6/c1-5-9-20-16(23)14-15(28-14)17(24)21-13(11(3)6-2)18(25)22-10-7-8-12(22)19(26)27-4/h11-15H,5-10H2,1-4H3,(H,20,23)(H,21,24)/t11-,12-,13-,14-,15-/m0/s1

methyl (2S)-1-[(2S,3S)-3-methyl-2-[[(2S,3S)-3-(propylcarbamoyl)oxirane-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carboxylate

Ca-074Me; Ca-074Me; CA-074 methyl ester; CA-074Me; Cathepsin B Inhibitor IV; methyl ((2S,3S)-3-(propylcarbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-prolinate; (S)-methyl 1-((2S,3S)-3-methyl-2-((2S,3S)-3-(propylcarbamoyl)oxirane-2-carboxamido)pentanoyl)pyrrolidine-2-carboxylate; methyl (2S)-1-[(2S,3S)-3-methyl-2-[[(2S,3S)-3-(propylcarbamoyl)oxirane-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carboxylate; CA-074-Me; Ca-074Me

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~251.59 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.29 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.29 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.29 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)