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]

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Adult male Sprague-Dawley rats (280-350 g) were used. Global cerebral ischemia was induced using the 4-vessel occlusion (4-VO) method. Both vertebral arteries were permanently electro-cauterized under anesthesia. The next day, both common carotid arteries were occluded with aneurysm clips for 20 minutes to induce ischemia, followed by clip removal for reperfusion. CA074-me was dissolved in 0.9% NaCl containing 2% DMSO to concentrations of 0.2 µg/µl or 2 µg/µl. One hour before ischemia or one hour post-reperfusion, 5 µl of the solution (containing 1 µg or 10 µg of CA074-me) or vehicle (2% DMSO) was injected into the right cerebral ventricle (coordinates: anteroposterior -0.92 mm; mediolateral 1.5 mm; dorsoventral 3.5 mm from bregma) at a rate of 0.5 µl/min [1].

[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 to CA-074 by cellular esterases. Many studies have shown that lysosomes play a crucial role in ischemic cell death in the brain, leading to the "lysosomal center" hypothesis. This hypothesis posits that altered or ruptured lysosomal membrane permeability (LMP) is essential for the release of cathepsin B, and inhibitors such as CA-074 and CA-074-me can prevent this process. However, the role of CA-074-me in neuronal death and its impact on altered lysosomal membrane integrity following global cerebral ischemia/reperfusion (I/R) injury remain unclear, hence our investigation. We assessed neuronal death in the CA1 region of the hippocampus in rats following 20 minutes of global cerebral I/R injury. CA074-me (1 μg and 10 μg) was administered intraventricularly 1 hour before ischemia or 1 hour after reperfusion. Changes in heat shock protein 70 (Hsp70), cathepsin B, lysosome-associated membrane protein 1 (LAMP-1), receptor-interacting protein 3 (RIP3), and lysosomal pH were assessed. CA074-me prevented programmed necrosis of neurons in the CA1 region induced by global cerebral ischemia/reperfusion injury both before and after ischemia/reperfusion injury. Two days after reperfusion, diffuse cytoplasmic cathepsin B and LAMP-1 immunostaining occurred simultaneously with nuclear pyknosis changes; after oxygen-glucose deprivation (OGD), lysosomal pH increased, accompanied by leakage of the lysosomal dye DND-153. Both changes indicate lysosomal membrane rupture and cathepsin B leakage, and CA074-me pretreatment significantly inhibited these changes. CA074-me pretreatment also inhibited RIP3 overexpression and nuclear translocation and the decrease in NAD(+) levels after ischemia/reperfusion injury, while enhancing Hsp70 upregulation. Delayed burst leakage of cathepsin B due to lysosomal rupture is a key detrimental factor in programmed neuronal necrosis induced by 20-minute global ischemia/reperfusion injury. In addition to acting as an inhibitor of cathepsin B, CA074-me may also exert an indirect neuroprotective effect by maintaining the integrity of the lysosomal membrane and preventing lysosomal rupture. [1] Osteoclasts are indispensable cells in the process of bone resorption. Since the occurrence of osteolytic diseases is closely related to the function and abnormalities of osteoclasts, understanding osteoclast biology is crucial for designing new therapies to inhibit osteolytic diseases. The identification and study of lysosomal proteases (such as cathepsins) have revealed the mechanisms of bone resorption. For example, cathepsin K has been shown to be a collagen-degrading protease produced by mature osteoclasts and has high activity in acidic osteoclast resorption pits. In-depth research on the mechanisms of action of cathepsins and other osteoclast-related compounds provides new targets for osteoclast biology research. Through our screening experiments on anti-osteoclastogenic compounds, we discovered a modified version of the cathepsin B inhibitor CA-074: the cell membrane-permeable CA-074Me (L-3-trans-(propylcarbamoyl)ethylene oxide-2-carbonyl]-L-isoleucyl-L-proline methyl ester). This study confirms that CA-074Me inhibits osteoclastogenesis in a dose-dependent manner both in vivo and in vitro. However, osteoclastogenesis in cathepsin B knockout mice was not affected, suggesting that its mechanism of action may be more complex than simply inhibiting cathepsin B. We found that CA-074Me exerts its osteoclastogenic inhibitory effect within 24 hours after osteoclastogenic stimulation by inhibiting the c-FOS and NFATc1 pathways. [2] Cathepsin B (CB) is involved in protein turnover and plays multiple roles in maintaining normal cellular metabolism. Our recent study found that CB expression is elevated in the muscles of patients with polymyositis/dermatomyositis (PM/DM). However, the role of CB in interstitial lung disease (ILD) has not been reported. Interstitial lung disease (ILD) is a common complication of polymyositis/diabetes mellitus (PM/DM) and a leading cause of death in PM/DM patients. ILD is a connective tissue disease with high morbidity and mortality, characterized by excessive production of inflammatory cytokines and induced fibrosis, ultimately leading to respiratory failure. The etiology and pathogenesis of ILD remain incompletely understood. This study aimed to investigate whether treatment with the CB-specific inhibitor CA-074Me could alleviate ILD symptoms in patients with polymyositis (PM). We analyzed CB expression, inflammation, and fibrosis in the lung tissue of PM/DM patients. A guinea pig PM animal model was induced using Coxsackievirus B1 (CVB1). CA-074Me was administered for 7 consecutive days, 24 hours after CVB1 injection. At the end of the experiment, animals were sacrificed and lung tissue was collected for further analysis. Inflammation, fibrosis, apoptotic cells, and cytokines were detected by histological examination, immunohistochemical analysis, Western blotting, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). In patients with multiple lung disease/dermatomyositis (PM/DM), the level of CB protein in lung tissue was significantly higher than that in healthy controls and was associated with increased inflammation and fibrosis. Similarly, in a Coxsackievirus B1 (CVB1)-induced guinea pig model of multiple lung disease (PM), CB expression, inflammation and fibrosis, CD8+ T cells, CD68+ cells, tumor necrosis factor-α, transforming growth factor-β1 infiltration, and apoptosis were all significantly increased in lung tissue. Administration of CA-074Me alleviated these changes. In summary, this study shows that PM/DM can increase CB expression in lung tissue, while inhibiting CB can alleviate interstitial lung disease (ILD) in a CVB1-induced PM guinea pig model. 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.)