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| Targets |
The primary target is cathepsin G, a serine protease secreted by activated neutrophils. No IC₅₀, Ki, EC₅₀, or DC₅₀ values were reported in this study. [1]
Chymostatin targets a broad range of proteases including α-, β-, γ-, δ-chymotrypsin, papain, chymases, cathepsins B and G, and lysosomal cysteine proteinases such as cathepsins A, B, C, H, and L. It shows Ki values of 9.36 nM for chymotrypsin, 13.1 nM for chymase, and 0.15 µM for cathepsin G. |
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| ln Vitro |
In cell-free enzymatic assays, chymostatin acts as a slow-binding, competitive inhibitor of chymotrypsin and chymase. The inhibition kinetics involve time-dependent inactivation in the presence of substrate. Ki values are determined from steady-state kinetic analyses. The compound consists of a mixture of type A (L-Leu), B (L-Val), and C (L-Ile) forms.
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| ln Vivo |
Chymostatin (2 mg/kg, i.p., administered 30 min before paraquat) significantly alleviated histopathological changes associated with acute alveolitis, including alveolar collapse, pulmonary haemorrhage, severe interstitial oedema, and massive neutrophil infiltration, compared to the paraquat group.
Chymostatin markedly decreased the lung wet‑to‑dry (W/D) ratio at 2‑48 h after treatment, indicating reduced pulmonary oedema. Chymostatin significantly decreased serum cathepsin G activity at 2‑48 h after treatment compared to the paraquat group. Chymostatin markedly and continuously increased serum endocan concentration at 2‑48 h after treatment compared to the paraquat group. Chymostatin efficiently decreased serum concentrations of TNF‑α, IL‑1β, and IL‑6 at 2‑48 h after treatment compared to the paraquat group. Chymostatin markedly decreased serum HMGB1 concentration at 8‑48 h after treatment compared to the paraquat group. Western blot analysis showed that Chymostatin up‑regulated endocan expression in lung tissues at 2‑48 h after treatment and down‑regulated nuclear NF‑κBp65 expression in lung tissues at 2‑48 h after treatment compared to the paraquat group. [1] Cellular studies use chymostatin as a protease inhibitor in lysis buffers at typical working concentrations of 6-60 µg/mL to prevent degradation of proteins during cell extraction. It is also used to study the role of chymotrypsin-like proteases in various cellular processes including angiotensin II regulation. |
| Enzyme Assay |
Cathepsin G enzymatic activity in serum was determined using the substrate Suc‑Phe‑Pro‑Phe‑pNA at pH 7.6. Activity was measured based on the quantity of p‑nitroaniline released after incubating the substrate (at a concentration of 74 mM) in serum for 2 hours at 37°C. Protein concentration was then determined using the biuret assay. [1]
The in vitro enzyme inhibition assay typically involves incubating purified chymotrypsin or chymase with varying concentrations of chymostatin (0.1 nM to 100 µM) and a fluorogenic or chromogenic substrate. The reaction is monitored continuously, and inhibition constants (Ki) are calculated from progress curves using non-linear regression. Assays are performed in buffer at pH 7.4-8.0 at 25-37°C. |
| Cell Assay |
Cells are lysed in buffers containing chymostatin at 6-60 µg/mL to preserve protein integrity. The compound is typically combined with other protease inhibitors such as leupeptin and pepstatin for complete proteolytic protection. Cells are incubated with the lysis buffer on ice for 30-60 minutes, followed by centrifugation and protein quantification.
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| Animal Protocol |
Animal model: Healthy female ICR mice (6‑8 weeks, 26‑30 g body weight) were used. Mice were randomly divided into five groups: NS group (0.9% saline, i.p.), DMSO group (0.05% DMSO, i.p.), Chymostatin group (Chymostatin in 0.05% DMSO at 2 mg/kg, i.p.), paraquat group (paraquat at 25 mg/kg, i.p.), and Chymostatin treatment group (Chymostatin in 0.05% DMSO at 2 mg/kg, i.p., 30 min before paraquat administration at 25 mg/kg, i.p.).
Six mice from each group were sacrificed at 0, 1, 2, 4, 8, 12, 24, and 48 h after treatment administration. Mice were anaesthetized with 0.3 mL/100 g of 10% chloral hydrate (i.p.). Blood samples were collected through cardiac puncture, centrifuged at 3000 rpm for 10 min at 4°C, and serum was stored at ‑80°C for analysis. Lung tissues were collected immediately after sacrifice. The superior lobe of the right lung was fixed in 10% paraformaldehyde, embedded in paraffin, sectioned at 5 µm thickness, and stained with haematoxylin and eosin (HE) for histopathological examination. The middle lobe of the right lung was used for W/D ratio determination (dried at 80°C for 72 h). The lower lobe of the right lung was frozen in liquid nitrogen for NF‑κBp65 Western blot analysis, and the lower lobe of the left lung was frozen for endocan Western blot analysis. [1] In vivo studies have been conducted in hypertensive rat models where chymostatin decreases plasma and tissue levels of angiotensin II without significantly lowering mean blood pressure. The compound is typically administered via intravenous injection or infusion. Endpoints include blood pressure measurement, renal hemodynamics assessment, and tissue angiotensin II quantification. |
| ADME/Pharmacokinetics |
Pharmacokinetic data for chymostatin are limited as it is primarily used as a research tool rather than a therapeutic agent. The peptide (C₃₁H₄₁N₇O₆, MW ~605) is soluble in DMSO (up to 20 mg/mL) and glacial acetic acid, but has limited aqueous solubility. It is stored as a powder at -20°C.
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| Toxicity/Toxicokinetics |
No specific toxicity data for Chymostatin were reported. However, the NS, DMSO, and Chymostatin groups showed normal lung histology with no histopathological changes, indicating no observable lung toxicity at the dose used (2 mg/kg). No other toxicity parameters were measured. [1]
Chymostatin is generally considered non-toxic at the concentrations used for protease inhibition in research applications (6-60 µg/mL). No significant cytotoxicity has been reported in standard cell culture applications. The compound is for research use only and is not intended for therapeutic use. |
| References | |
| Additional Infomation |
Chymostatin (CAS# 9076‑44‑2) is a cathepsin G inhibitor. This study demonstrates its protective effects against paraquat‑induced acute lung injury in mice by inhibiting cathepsin G activity, which leads to up‑regulation of endocan expression (by preventing its degradation by cathepsin G) and indirect inhibition of NF‑κBp65 activity. Endocan competes with ICAM‑1 for LFA‑1 binding, inhibiting leukocyte migration and infiltration. Cathepsin G degrades endocan from a 50 kDa intact protein into 14 kDa fragments. By inhibiting cathepsin G, Chymostatin preserves endocan levels, reducing leukocyte‑mediated lung injury. Additionally, cathepsin G can activate NF‑κB by degrading phospholipid transfer protein; thus, Chymostatin indirectly inhibits NF‑κB activation, reducing pro‑inflammatory cytokine (TNF‑α, IL‑1β, IL‑6, HMGB1) production. This study provides evidence that cathepsin G is a potential therapeutic target for acute lung injury and that Chymostatin may be a promising therapeutic agent for paraquat poisoning. No FDA approval or clinical use information is mentioned. [1]
Chymostatin was first discovered as a new chymotrypsin inhibitor produced by actinomycetes in 1970. It is a mixture of three structural variants A (L-Leu), B (L-Val), and C (L-Ile). The compound has been used extensively as a research tool for studying serine protease function and as a component of protease inhibitor cocktails for protein purification. |
| Molecular Formula |
C31H41N7O6
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|---|---|
| Molecular Weight |
607.71
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| Exact Mass |
607.311
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| CAS # |
9076-44-2
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| PubChem CID |
443119
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Melting Point |
276-278℃
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| Index of Refraction |
1.640
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| LogP |
4
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| Hydrogen Bond Donor Count |
7
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
15
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| Heavy Atom Count |
44
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| Complexity |
1010
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| Defined Atom Stereocenter Count |
4
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| SMILES |
CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C=O)NC(=O)[C@H](C2CCN=C(N2)N)NC(=O)N[C@@H](CC3=CC=CC=C3)C(=O)O
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| InChi Key |
MRXDGVXSWIXTQL-HYHFHBMOSA-N
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| InChi Code |
InChI=1S/C31H41N7O6/c1-19(2)15-24(27(40)34-22(18-39)16-20-9-5-3-6-10-20)35-28(41)26(23-13-14-33-30(32)36-23)38-31(44)37-25(29(42)43)17-21-11-7-4-8-12-21/h3-12,18-19,22-26H,13-17H2,1-2H3,(H,34,40)(H,35,41)(H,42,43)(H3,32,33,36)(H2,37,38,44)/t22-,23?,24-,25-,26-/m0/s1
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| Chemical Name |
(2S)-2-[[(1S)-1-(2-amino-1,4,5,6-tetrahydropyrimidin-6-yl)-2-[[(2S)-4-methyl-1-oxo-1-[[(2S)-1-oxo-3-phenylpropan-2-yl]amino]pentan-2-yl]amino]-2-oxoethyl]carbamoylamino]-3-phenylpropanoic acid
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| Synonyms |
DB01683 AM-037855DB-01683 AM037855DB 01683
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~50 mg/mL
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (Infinity 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. Solubility in Formulation 2: 2.5 mg/mL (Infinity mM) in 10% DMSO + 90% (20% SBE-β-CD in 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 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6455 mL | 8.2276 mL | 16.4552 mL | |
| 5 mM | 0.3291 mL | 1.6455 mL | 3.2910 mL | |
| 10 mM | 0.1646 mL | 0.8228 mL | 1.6455 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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