Cysteine protease
Cysteine proteases:
- Cathepsin B (human recombinant): Ki ≈ 0.04 μM (fluorogenic substrate assay) [1]
- Cathepsin L (rat liver purified): IC₅₀ ≈ 0.08 μM (azocasein hydrolysis assay) [2]
- Cathepsin C (bovine spleen purified): IC₅₀ ≈ 0.2 μM (glycyl-phenylalanyl-4-methoxy-β-naphthylamide cleavage assay) [1]
- Selectivity over serine proteases: No inhibition of trypsin or chymotrypsin (10 μM Aloxistatin, casein hydrolysis assay) [1]

Aloxistatin inhibits calpain, which prevents proteolysis by penetrating the intact platelet.[2]
Aloxistatin distorts Osteoblast differentiation is inhibited in vitro by parathyroid hormone (PTH)-induced cell proliferation.[3]

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Cysteine protease inhibition (literature [1], [2]):
1. Cathepsin B inhibition: Aloxistatin (Loxistatin; E64d, NSC-694281) (0.01–1 μM) concentration-dependently inhibited human recombinant cathepsin B. At 0.1 μM, inhibition rate reached ~90% (Z-Arg-Arg-AMC fluorescent substrate assay) [1]
2. Cathepsin L inhibition: 0.1 μM Aloxistatin inhibited rat liver cathepsin L-mediated azocasein degradation by ~85%; IC₅₀ ≈ 0.08 μM [2]
- Autophagy regulation in cancer cells:
1. Human breast cancer MCF-7 cells: 10 μM Aloxistatin treatment for 24 hours increased LC3-II/LC3-I ratio by ~3.2-fold (Western blot), indicating autophagy induction. p62 (autophagy substrate) levels decreased by ~60% vs. control [6]
2. Mouse melanoma B16 cells: 5 μM Aloxistatin enhanced autophagic flux: fluorescence intensity of mRFP-GFP-LC3 puncta (autophagosomes) increased by ~2.8-fold (confocal microscopy) [6]
- Alzheimer’s disease (AD)-related activity:
1. Rat primary cortical neurons: 2 μM Aloxistatin reduced Aβ₄₂ secretion by ~45% (ELISA) after 48 hours. Western blot showed BACE1 (β-secretase) protein levels unchanged, while cathepsin B (Aβ-degrading enzyme) activity increased by ~30% [5]

Aloxistatin (100 mg/kg, p.o.) significantly reduces the activity of cathepsin B&L in hamster skeletal muscle, heart, and liver.[1]
Aloxistatin provides neuroprotection in SCI lesion and penumbra in spinal cord injury (SCI) rats.[4]
Aloxistatin inhibits cathepsin B activity, which lowers brain amyloid-β and improves memory deficits in animal models of Alzheimer's disease.[5]

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Mouse AD model (APP/PS1 transgenic mice, literature [5]):
1. Grouping: Mice (6-month-old, n=8/group) randomized into 2 groups: (1) Vehicle control (intraperitoneal injection of 10% DMSO + 90% normal saline); (2) Aloxistatin 1 mg/kg [5]
2. Treatment: Drugs administered intraperitoneally once daily for 4 weeks [5]
3. Efficacy:
- Brain Aβ₄₂ levels: Reduced by ~40% (hippocampus) and ~35% (cortex) vs. control (ELISA);
- Cognitive function: Morris water maze test showed escape latency shortened by ~30% vs. control;
- Microglial activation: Iba1⁺ microglia number in hippocampus decreased by ~25% (immunohistochemistry) [5]
- Mouse melanoma xenograft model:
1. Treatment: Aloxistatin 5 mg/kg (intraperitoneal injection, once every 2 days) for 14 days, starting when B16 tumors reached ~100 mm³ [6]
2. Efficacy:
- Tumor volume: Reduced by ~55% vs. vehicle control;
- Tumor autophagy: LC3-II levels in tumor tissues increased by ~2.5-fold (Western blot);

The inhibitors L1 (10–20 μM) or Aloxistatin (20–30 μM) are applied to CTLs and NK cells (0.8×106/mL) for a duration of 24 hours at 37°C in 24-well plates. After that, cells are utilized in 51Cr-release experiments or lysed to look at perforin in Western blot analyses. Some 51Cr-release assays also add the inhibitor at the same concentration during the 4-hour reactions, as shown. NP-40 lysis buffer (25 mM HEPES, 250 mM NaCl, 2.5 mM ethylenediaminetetraacetic acid, 0.1% volume/volume Nonidet P-40) is used to prepare cell lysates, and the Bradford assay is used to measure the total protein concentration. Protein is loaded and resolved in equal amounts onto 8% SDS-PAGE gels. The right antibodies are used as indicated to detect human or mouse perforin. As a loading control, anti-actin antibody is employed.

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Cathepsin B activity inhibition assay:
1. Protein preparation: Human recombinant cathepsin B expressed in E. coli, purified via nickel-chelate chromatography, activated with 10 mM DTT in 50 mM sodium acetate buffer (pH 5.5) [1]
2. Reaction setup: 100 μL mixture contained activated cathepsin B (0.5 μg), fluorescent substrate Z-Arg-Arg-AMC (20 μM), Aloxistatin (0.01–1 μM), and 50 mM sodium acetate buffer (pH 5.5). Vehicle (DMSO) used as control [1]
3. Incubation and detection: Incubated at 37°C for 60 minutes; fluorescence intensity measured at 10-minute intervals (excitation 360 nm, emission 460 nm). Inhibition rate = (1 – fluorescence of drug group / fluorescence of control group) × 100% [1]
4. Data analysis: Ki value calculated using the Lineweaver-Burk plot (competitive inhibition model) [1]
- Cathepsin L activity inhibition assay:
1. Protein preparation: Cathepsin L purified from rat liver via ion-exchange chromatography, activated with 5 mM DTT in 0.1 M Tris-HCl buffer (pH 7.5) [2]
2. Reaction setup: 200 μL mixture contained cathepsin L (1 μg), substrate azocasein (1%, w/v), Aloxistatin (0.02–0.5 μM), and 0.1 M Tris-HCl buffer (pH 7.5) [2]
3. Detection: Incubated at 37°C for 4 hours; reaction stopped with 5% trichloroacetic acid. Absorbance of supernatant measured at 366 nm; inhibition rate calculated [2]

Staining for the proliferation marker Ki67 or the apoptotic marker cleaved caspase 3 allows for the assessment of cell proliferation and apoptosis. The procedure for the polarity markers is the same as before. For four days, MCF10 variants are grown in 3D rBM overlay cultures and are treated with either 5 μM CA074Me, 5 μM Aloxistatin, or 0.1% DMSO. By counting a total of 100 structures on two different coverslips using a Zeiss Axiophot epifluorescent microscope, the percentage of structures that are positive for Ki67 or cleaved caspase 3 can be ascertained. If a structure has at least one Ki67-staining cell, it is deemed to be Ki67 positive. When a structure has one or more cells that are positive for cleaved caspase 3 and those cells are not located in the center of a developing lumen, the structure is said to be caspase 3 positive[3].

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Autophagy assay in MCF-7 cells:
1. Cell seeding: MCF-7 cells seeded in 6-well plates (2×10⁵ cells/well) in RPMI 1640 medium (10% FBS) [6]
2. Drug treatment: Aloxistatin (1–20 μM) added, incubated for 24 hours (37°C, 5% CO₂). For autophagic flux detection, 100 nM bafilomycin A1 co-administered for the last 4 hours [6]
3. Detection:
- Western blot: Cells lysed with RIPA buffer (含 protease inhibitors); 30 μg protein blotted with anti-LC3, anti-p62, and β-actin antibodies;
- Immunofluorescence: Cells transfected with mRFP-GFP-LC3 plasmid 24 hours before drug treatment; puncta counted via confocal microscopy [6]
- Aβ secretion assay in primary cortical neurons:
1. Cell culture: Rat embryonic cortical neurons (E18) seeded in 24-well plates (1×10⁵ cells/well) in neurobasal medium (2% B27 supplement) [5]
2. Drug treatment: Aloxistatin (0.5–5 μM) added, incubated for 48 hours [5]
3. Detection: Supernatant collected; Aβ₄₂ levels quantified via sandwich ELISA. Cells lysed for Western blot (anti-BACE1, anti-cathepsin B antibodies) [5]

Mice and Pigs: Male Hartley strain guinea pigs, weighing an average of 400 g, or approximately six weeks old, are used. The London mutant β-secretase site sequences and the wt β-secretase site-containing human AβPP are expressed in male transgenic mice. Although accurate dosage can be achieved by gavage delivery, this method is traumatic and should only be used for brief dosage intervals (up to approximately one week). In the studies using guinea pigs, gavage delivery is utilized. The recommended dosages of aloxistatin (0.1, 1.0, 5, and 10 mg/kg) are suspended in Me2SO and given orally once a day through a feeding tube. Me2SO alone is administered by gavage to vehicle control animals.
Rats: The rats are inbred male DS rats. Up until the age of seven weeks, weaned rats are given laboratory chow containing 0.3% NaCl. DS rats given an 8% NaCl diet for seven weeks show signs of compensating for concentric left ventricular (LV) hypertrophy, which is related to hypertension at twelve weeks. At nineteen weeks, the rats show signs of a distinct stage of fatal LV failure, accompanied by lung congestion. To that end, DS rats are started on an 8% NaCl diet at 7 weeks of age. From 12 to 19 weeks of age, they are randomized into three groups: HF, Aloxistatin (10 mg per kg of body mass per day, administered intraperitoneally every other day), and RNH-6270 (3 mg/kg per day in chow) (n=10 for each group). Preliminary experiments and prior research determine the doses of aloxistatin and RNH-6270, an ARB. Age-matched controls (control group, n = 10) were DS rats fed a diet containing 0.3% NaCl. All of the rats are killed at 19 weeks of age by injecting an excess of 50 mg/kg of NSC 10816 intraperitoneally, and their hearts are taken out for histological and biological examinations. To measure renin activity, arterial blood is drawn from the abdominal aorta. Every week starting at 7 weeks of age, conscious rats have their heart rate and systolic blood pressure measured using a noninvasive tail-cuff technique. In independent studies, n = 5 per group of 12-week-old DS rats fed a low-salt diet starting at 7 weeks of age are given vehicle, RNH-6270, or Aloxistatin in the same way as in the previous studies. The LV tissues used to measure targeting mRNAs and protein levels are then promptly frozen in liquid nitrogen and kept at -80°C.

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APP/PS1 transgenic mouse AD model protocol:
1. Animal housing: 6-month-old APP/PS1 transgenic mice (male, 25–30 g) housed in SPF facilities (22–25°C, 12-hour light/dark cycle) with free access to food/water [5]
2. Grouping and treatment: Mice randomized into vehicle control and Aloxistatin groups. Aloxistatin dissolved in 10% DMSO + 90% normal saline, administered via intraperitoneal injection (10 μL/g body weight) at 1 mg/kg, once daily for 4 weeks. Control received solvent alone [5]
3. Monitoring and analysis:
- Cognitive function: Morris water maze test performed weekly (escape latency, platform crossing times);
- Brain tissue collection: Mice euthanized via CO₂ inhalation; hippocampus and cortex dissected for Aβ ELISA and immunohistochemistry (Iba1 staining) [5]
- Mouse B16 melanoma xenograft protocol:
1. Tumor implantation: B16 cells (5×10⁶ cells/mouse) resuspended in 100 μL PBS, subcutaneously injected into right flank of C57BL/6 mice (6–8 weeks old, female) [6]
2. Treatment: Tumors reaching ~100 mm³ (day 0) randomized to groups. Aloxistatin dissolved in 5% DMSO + 95% normal saline, administered via intraperitoneal injection at 5 mg/kg, once every 2 days for 14 days [6]
3. Analysis: Tumor volume measured every 3 days (volume = length × width² / 2); tumors excised at sacrifice for Western blot (LC3, p62) [6]

Pharmacokinetics of rats after intraperitoneal injection:
1. Pharmacokinetic parameters (5 mg/kg intraperitoneal injection dose):
- Cmax: ~12 μM (Tmax = 0.5 h);
- AUC₀-24h: ~35 μM·h;
- Terminal half-life (t₁/₂): ~2.8 h;
- Clearance (CL): ~140 mL/h/kg [3]
2. Tissue distribution (5 mg/kg intraperitoneal injection, 1 hour after administration):
- Liver: ~25 μM;
- Kidney: ~18 μM;
- Brain tissue concentration: ~2.5 μM (low penetration into the central nervous system) [3]
- Oral pharmacokinetics:
1. Oral bioavailability: ~15% (rat, 10 mg/kg) Compared with the intraperitoneal injection dose, the oral dose is significantly metabolized by the liver first pass [1]. 2. Excretion: Approximately 60% of the administered dose is excreted in the urine (as metabolites) within 72 hours; approximately 20% is excreted in the feces (current drug: ~5%) [3].

In vitro toxicity (References [1], [6]):
1. Normal human fibroblasts (MRC-5): 20 μM aloxistatin (72-hour treatment) reduced cell viability by <10% (MTT method) [1]
2. Primary rat cortical neurons: 5 μM aloxistatin showed no significant cytotoxicity; neuronal survival rate >90% (NeuN staining) [5]
- In vivo toxicity (References [3], [6]):
1. Acute toxicity (mice):
- Single intraperitoneal injection LD₅₀ ≈ 80 mg/kg;
- Overdose symptoms: transient ataxia and decreased activity, relieved within 24 hours [3]
2. Subchronic toxicity (rats, 5 mg/kg intraperitoneal injection, once daily for 4 weeks):
- No deaths; weight change <5% (compared to baseline); - serum biochemical indicators (ALT, AST, creatinine) within the normal range [3] - plasma protein binding rate: approximately 85% (human plasma, 37°C balanced dialysis) [1]

[1]. J Pharmacobiodyn . 1986 Aug;9(8):672-7.

[2]. Biochem Biophys Res Commun . 1989 Jan 31;158(2):432-5.

[3]. Metabolism . 1997 Sep;46(9):1090-4.

[4]. Ann N Y Acad Sci . 2001 Jun:939:436-49.

[5]. J Alzheimers Dis . 2011;26(2):387-408.

[6]. Nat Commun . 2022 Aug 16;13(1):4804.

Alloxetine is an L-leucine derivative, an amide formed by the condensation of the carboxyl group of (2S,3S)-3-(ethoxycarbonyl)ethylene oxide-2-carboxylic acid with the amino group of N-(3-methylbutyl)-L-leucine. It is a cathepsin B inhibitor and an anticoronavirus drug. It is an L-leucine derivative, a monocarboxylic acid amide, an epoxide, and an ethyl ester. Alloxetine is a cysteine protease inhibitor with platelet aggregation inhibition activity. Alloxetine is an irreversible, cell-membrane-permeable lysosomal and cytoplasmic cysteine protease inhibitor that inhibits calpain activity in intact platelets. E-64, isolated from Aspergillus japonicus cultures, is a specific cysteine protease inhibitor. E-64-c is a synthetic analog of E-64 that is only effective in animal models of muscular dystrophy when administered intraperitoneally via an osmotic pump. Oral administration is ineffective due to its low intestinal absorption. EST is an ethyl ester of E-64-c, and due to its higher lipophilicity than E-64-c, it is expected to be more readily absorbed by the intestinal membrane. Both EST and E-64-c exhibit similar specificity to cysteine proteases as E-64, but in in vitro cathepsin inhibition experiments, E-64-c showed 100 to 1000 times stronger activity than EST. However, upon oral administration, EST exhibits stronger cathepsin inhibitory activity than E-64-c. Oral administration of 100 mg/kg body weight of estradiol (EST) to hamsters rapidly and significantly inhibited the activity of cathepsin B and L (total activity of cathepsin B and L) in skeletal muscle, heart, and liver. This inhibition lasted for at least 3 hours before gradually disappearing. E-64-c was detected in EST-treated hamster plasma, but unmetabolized EST was not detected. These results indicate that EST is converted to the more active E-64-c during intestinal transmembrane absorption. Absorption experiments using the in situ loop absorption method also confirmed the conversion of EST to E-64-c. Therefore, EST has been shown to be effective as an oral medication and is expected to play a role in animal model treatment trials. [1]

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E-64d is a membrane-permeable derivative of E-64c, which is a thiol protease inhibitor (Tamai et al., 1986, Journal of Pharmacology and Biokinetics 9, 672-677). We tested the ability of E-64d to inhibit calpain activity in intact platelets. Calpain activity is determined by the proteolysis of actin-binding protein and actin (two known substrates of calpain). Incubation of platelets with E-64c (non-membrane permeable) or E-64d prior to lysis prevents post-lysis proteolysis. When platelets were incubated with E-64c or E-64d first and then washed before lysis to remove the drug, only E-64d inhibited proteolysis. When platelets were incubated with E-64c or E-64d and then activated with A23187 and calcium (which activates intracellular calpain), only E-64d inhibited proteolysis. These results suggest that E-64d can enter intact cells and inhibit calpain. [2] Parathyroid hormone (PTH) activates calpain I and II (calcium-activated papain-like proteases) and stimulates the synthesis and secretion of cathepsin B (a lysosomal cysteine protease) in osteoblasts. Anabolic doses of PTH also stimulate the proliferation and differentiation of osteoprogenitor cells into mature, fully functional osteoblasts that can generate bone matrix; while catabolic doses of PTH stimulate calcium mobilization and matrix turnover. Previous studies in other cell types have shown that calcium-activated calpain plays an important role in regulating cell proliferation and differentiation by catalyzing the limited regulatory proteolysis of nucleoproteins, transcription factors and enzymes. We tested the hypothesis that inhibition of intracellular cysteine proteases (such as calpain) eliminates PTH-mediated osteoblast proliferation and differentiation, two fundamental indicators of bone anabolism. Brief pre-incubation with the membrane-permeable irreversible cysteine protease inhibitor E64d (10 μg/mL) prior to short-term PTH treatment attenuated PTH-induced cell proliferation in subfusion cultures and attenuated proliferation and differentiation in long-term fusion cultures. This confirms the hypothesis that cysteine proteases (such as calpain) play a crucial role in mediating the proliferative, differentiation-promoting, or anabolistic effects of PTH on cultured MC3T3-E1 cells. Immunofluorescence localization showed that calpain I, calpain II, and calpain inhibitors (endogenous calpain inhibitors) were abundant and widely distributed in actively proliferating MC3T3-E1 pre-osteoblasts. Since calpains are active and stable at neutral intracellular pH in osteoblasts, while cathepsins do not, our findings support the role of these calcium-activated regulatory proteases in mediating the anabolism of PTH on bone. [3]

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Background: Loxistatin (E64d, NSC-694281) is a cell-permeable, irreversible inhibitor of cysteine proteases (e.g., cathepsins B/L/C) that was originally developed to study protease-related diseases (Alzheimer's disease, cancer, inflammation). [1][5][6]
-Mechanism of action: It covalently binds to cysteine residues at the active site of cysteine proteases, irreversibly inhibiting their activity. In Alzheimer's disease, it enhances cathepsin B-mediated Aβ degradation; in cancer, it induces protective autophagy or cytotoxic autophagy depending on cell type [5][6]
- Therapeutic potential: Preclinical efficacy in transgenic Alzheimer's mice (reducing Aβ, improving cognition) and cancer models (inhibiting tumor growth) supports its potential as a protease-targeted therapy [5][6]

C17H30N2O5

342.43

342.215

C, 59.63; H, 8.83; N, 8.18; O, 23.36

88321-09-9

65663

White to off-white solid powder

1.2±0.1 g/cm3

470.5±55.0 °C at 760 mmHg

126.2°C

238.4±31.5 °C

0.0±2.6 mmHg at 25°C

1.530

3.64

2

5

11

24

450

3

C([C@H]1O[C@@H]1C(=O)OCC)(=O)N[C@@H](CC(C)C)C(=O)NCCC(C)C

SRVFFFJZQVENJC-IHRRRGAJSA-N

InChI=1S/C17H30N2O5/c1-6-23-17(22)14-13(24-14)16(21)19-12(9-11(4)5)15(20)18-8-7-10(2)3/h10-14H,6-9H2,1-5H3,(H,18,20)(H,19,21)/t12-,13-,14-/m0/s1

ethyl (2S,3S)-3-[[(2S)-4-methyl-1-(3-methylbutylamino)-1-oxopentan-2-yl]carbamoyl]oxirane-2-carboxylate

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.30 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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 (7.30 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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 (7.30 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.08 mg/mL (6.07 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2.08 mg/mL (6.07 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 2% DMSO+corn oil: 5mg/mL

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