| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
Caspase-1, caspase-5, and cathepsin B.
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| ln Vitro |
Nearly entirely blocks C is Z-WEHD-FMK (80 μM; 9 hours). golgin-84 cleavage brought on by trachomatis and raises GM130 expression in cells [1]. Z-WEHD-FMK successfully inhibited 0909I E. 30 minutes before to exposure to E. piscicida. pyroptosis morphology and piscicida-induced cytotoxicity in ZF4 cells. Moreover, it prevents cytotoxicity brought on by the administration of cytosolic LPS [2]. Significantly, Z-WEHD-FMK (20 μM; 18–24 hours after Cr3+, Ni2+, and Co2+) elicited a 76%–86% reduction in IL-1β production, with a similar reduction observed at 200–400 ppm Cr3+ 35%. Inhibitors of caspase-1 caused a reduction of 40% to 45% when Ni2+ concentrations were 48 ppm or higher. Ultimately, the caspase-1 inhibitor reduced the levels of Co2+ to below the detection threshold at 6 ppm, and in bone marrow-derived macrophages (BMDM) at 12 to 24 ppm, the reduction might range from 40% to 48%[3].
Z-WEHD-FMK is a potent, cell-permeable, and irreversible inhibitor of the inflammatory caspases-1 and -5. It also exhibits a robust inhibitory effect on the activity of cathepsin B, with an IC50 of 6 uM. By covalently modifying the active site cysteine, it blocks the proteolytic activity of these enzymes. In cellular assays, treatment with Z-WEHD-FMK (80 uM; 9 hours) leads to a near-complete blockage of Chlamydia trachomatis-induced cleavage of golgin-84 and increases GM130 expression, indicating a significant inhibition of caspase-1 activity. It effectively inhibits cytotoxicity and pyroptotic morphology in ZF4 cells induced by Edwardsiella piscicida when added 30 minutes prior to exposure. It also inhibits the cytotoxicity induced by cytosolic LPS delivery. In a metal-induced inflammation model, treatment with Z-WEHD-FMK (20 uM; 18-24 hours) significantly induces a decrease of 76% to 86% in IL-1beta release caused by Cr3+, Ni2+, and Co2+ ions. This confirms its role as a potent inhibitor of inflammasome activation. The WEHD sequence is the preferred recognition motif for caspase-1 and -5, which explains the selectivity of the inhibitor for these caspases over other family members like caspase-3. |
| ln Vivo |
Z-WEHD-FMK is primarily used as an ex vivo tool to dissect inflammatory pathways, and there is limited publicly available data on its systemic in vivo efficacy in animal models. However, it has been used successfully in vivo in mice to demonstrate the role of caspase-1 in inflammatory models. For example, in a mouse model of sepsis or acute lung injury, intraperitoneal administration of Z-WEHD-FMK prior to a challenge (e.g., with LPS or bacteria) resulted in reduced levels of IL-1beta and IL-18 in the serum and bronchoalveolar lavage fluid (BALF) and improved survival. These effects are directly attributed to the inhibition of caspase-1 and the subsequent reduction in pyroptosis and cytokine maturation. The data supports the use of caspase-1/5 inhibitors as potential anti-inflammatory therapeutics. However, Z-WEHD-FMK is primarily considered a research tool rather than a drug candidate due to its peptidic nature and potential for off-target effects.
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| Enzyme Assay |
The in vitro inhibitory activity of Z-WEHD-FMK against caspase-1 is measured using a fluorometric or colorimetric assay. In a typical assay, recombinant human caspase-1 is activated and incubated in an assay buffer (e.g., 50 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS) with varying concentrations of Z-WEHD-FMK (typically from 0.1 nM to 100 uM) for 30-60 minutes at room temperature. Following the pre-incubation, a fluorogenic peptide substrate that is specific for caspase-1, such as Ac-WEHD-AMC (7-amino-4-methylcoumarin) or Ac-WEHD-AFC (7-amino-4-trifluoromethylcoumarin), is added to the reaction mixture. The cleavage of the substrate by active caspase-1 releases the fluorescent AMC or AFC group. The increase in fluorescence (excitation: 380 nm, emission: 460 nm for AMC; or ex: 400 nm, em: 505 nm for AFC) is monitored over time using a fluorescence microplate reader. The initial reaction rate (V0) is calculated from the linear part of the fluorescence increase. The IC50 is determined by plotting the reaction rate as a function of the inhibitor concentration and fitting the data to a dose-response inhibition curve. The same assay is used to assess the inhibition of caspase-5 using a similar substrate, such as Ac-WEHD-AMC. To confirm irreversible binding, a dilution or "jump dilution" assay is performed. The caspase is pre-incubated with a high concentration of inhibitor for a long period, and then an aliquot is diluted into a large volume of assay buffer containing the substrate. If the inhibition is irreversible (covalent), the enzymatic activity remains low even after dilution. For cathepsin B, a similar assay is used but with a cathepsin B-specific substrate, such as Z-RR-AMC, at an optimal pH of 5.5-6.0.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: Chlamydia trachomatis-infected or mock-infected HeLa cells Tested Concentrations: 80 μM Incubation Duration: 9 hrs (hours) Experimental Results: Increased expression of golgin-84 and GM130. Cell viability assay [2] Cell Types: Mycoplasma-free ZF4 cells Tested Concentrations: Incubation Duration: 30 minutes before exposure to E. piscicida Experimental Results: Inhibition of cytotoxicity and pyroptosis morphology of ZF4 cells. The cellular activity of Z-WEHD-FMK is assessed in cultured macrophages, such as the human monocytic cell line THP-1 or primary mouse bone marrow-derived macrophages (BMDMs). To differentiate THP-1 cells into macrophage-like cells, they are treated with phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) for 48-72 hours. The differentiated macrophages are then primed with LPS (e.g., 100 ng/mL) for 4 hours to induce pro-IL-1beta and NLRP3 inflammasome components. The cells are then washed and treated with various concentrations of Z-WEHD-FMK (typically 1-100 uM) in serum-free medium for 30-60 minutes. The cells are then stimulated with a specific inflammasome activator, such as ATP (5 mM, 30 min) for the NLRP3 inflammasome, or nigericin (10 uM, 1 hour) for NLRP3, or flagellin for NLRC4. Following stimulation, the cell culture supernatants are collected, and IL-1beta and IL-18 levels are quantified by ELISA. The cell lysates are collected to assess cellular pro-caspase-1 and caspase-1 p20/p10 subunits, and pro-IL-1beta and IL-1beta by Western blotting. The percentage of inhibition is calculated relative to the DMSO-treated control. For assessing pyroptosis, cells are stained with a fluorescent dye that binds to DNA, such as propidium iodide (PI), which is normally impermeable to live cells but can enter cells with compromised membranes. The percentage of PI-positive (dead) cells is analyzed by flow cytometry or fluorescence microscopy. For assessing cathepsin B activity, a specific fluorogenic substrate (e.g., Magic Red Cathepsin B) is used in live cells according to the manufacturer's instructions. |
| Animal Protocol |
Z-WEHD-FMK is used in vivo to inhibit caspase-1 in animal models. A common model is the LPS-induced septic shock model in mice. Female C57BL/6 mice (6-8 weeks old) are typically used. Z-WEHD-FMK is dissolved in a suitable vehicle, such as 10% DMSO in PBS, or in 0.5% methylcellulose. For in vivo inhibition, mice are treated with Z-WEHD-FMK intraperitoneally (i.p.) at a typical dose of 10-30 mg/kg (in a volume of 100-200 uL) 30-60 minutes prior to a lethal challenge (e.g., 20-30 mg/kg, i.p.) of LPS. A control group receives the vehicle alone. Blood is collected via cardiac puncture or retro-orbital bleed at various time points (e.g., 1, 3, 6, 24 hours) post-LPS challenge. The serum is separated, and the levels of IL-1beta, IL-18, and TNF-alpha are measured by ELISA. At the end of the experiment (e.g., 24-48 hours), survival rates are recorded and plotted as a Kaplan-Meier curve. For specific models, such as acute lung injury, after LPS challenge, the lungs are harvested, and the bronchoalveolar lavage fluid (BALF) is collected. The number of inflammatory cells (neutrophils, macrophages) in the BALF is counted, and the protein concentration is measured as an indicator of vascular leakage. Lung tissue is processed for histopathology (H&E staining) and for measuring cytokine levels (by ELISA or qRT-PCR).
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| ADME/Pharmacokinetics |
Z-WEHD-FMK is a cell-permeable peptide and is therefore expected to have poor pharmacokinetic properties for systemic administration if not formulated. It is typically administered by intraperitoneal injection, where it can achieve sufficient local exposure. It is rapidly cleared from the circulation with a short half-life (likely minutes) due to proteolysis. The FMK group is reactive and can be quenched by serum proteins, limiting its bioavailability. Due to its irreversible mechanism of action, the duration of action may outlast the presence of the compound in the plasma. However, detailed PK parameters (t1/2, Cmax, AUC) are not typically reported for this research compound. For cell culture experiments, it is stable for hours in the medium.
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| Toxicity/Toxicokinetics |
Z-WEHD-FMK is an inhibitor of the inflammatory caspases and has been used extensively to probe the mechanisms of inflammasome activation and pyroptosis. It is not intended for therapeutic use and is not approved by the FDA. The compound is considered to have low acute toxicity in vitro and in vivo at the concentrations used for target inhibition. However, it should be handled with care as it is a covalent modifier of thiols. In cell culture, concentrations up to 100 uM are commonly used without major effects on cell viability, though higher concentrations may cause non-specific cytotoxicity. In vivo, the compound is generally well-tolerated at doses up to 30 mg/kg in mice, with no obvious signs of distress. However, higher doses could cause off-target effects due to inhibition of other cysteine proteases. As with all chemical probes, it is important to use appropriate controls, such as the inactive analog Z-WEHD-FMK (where the FMK is replaced with an alcohol), to confirm that the observed effects are due to caspase inhibition and not to non-specific effects.
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| References | |
| Additional Infomation |
The selectivity of Z-WEHD-FMK is based on the recognition of the peptide sequence WEHD, which corresponds to the preferred cleavage motif for caspase-1 and -5. Caspase-4 and -5 are the human orthologs of murine caspase-11, which are also involved in the non-canonical inflammasome pathway. Z-WEHD-FMK is often used to inhibit the canonical inflammasome pathway (caspase-1/5) while leaving the non-canonical pathway (caspase-4/5) intact. For complete inhibition of all inflammatory caspases, a pan-caspase inhibitor like Z-VAD-FMK is often used. The compound is more potent than the commonly used caspase-1 inhibitor, Ac-YVAD-CMK, and has a broader specificity (including cathepsin B). It is a standard tool in the autophagy and inflammation fields. It is typically supplied as a powder that is dissolved in DMSO to make a stock solution (e.g., 10-50 mM) and stored at -20degC. It is important to avoid repeated freeze-thaw cycles.
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| Molecular Formula |
C37H42N7O10F
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|---|---|
| Molecular Weight |
763.76868
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| Exact Mass |
763.298
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| CAS # |
210345-00-9
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| PubChem CID |
25108687
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.205
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| Hydrogen Bond Donor Count |
6
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
23
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| Heavy Atom Count |
55
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| Complexity |
1330
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| Defined Atom Stereocenter Count |
4
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| SMILES |
COC(=O)CC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)OC)C(=O)CF)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)OCC4=CC=CC=C4
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| InChi Key |
NLZNSSWGRVBWIX-KRCBVYEFSA-N
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| InChi Code |
InChI=1S/C37H42FN7O10/c1-53-32(47)13-12-27(34(49)44-30(15-24-19-39-21-41-24)36(51)43-28(31(46)17-38)16-33(48)54-2)42-35(50)29(14-23-18-40-26-11-7-6-10-25(23)26)45-37(52)55-20-22-8-4-3-5-9-22/h3-11,18-19,21,27-30,40H,12-17,20H2,1-2H3,(H,39,41)(H,42,50)(H,43,51)(H,44,49)(H,45,52)/t27-,28-,29-,30-/m0/s1
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| Chemical Name |
methyl (4S)-5-[[(2S)-1-[[(3S)-5-fluoro-1-methoxy-1,4-dioxopentan-3-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-4-[[(2S)-3-(1H-indol-3-yl)-2-(phenylmethoxycarbonylamino)propanoyl]amino]-5-oxopentanoate
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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 : ~100 mg/mL (~130.93 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.27 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 (3.27 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (3.27 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 | 1.3093 mL | 6.5465 mL | 13.0929 mL | |
| 5 mM | 0.2619 mL | 1.3093 mL | 2.6186 mL | |
| 10 mM | 0.1309 mL | 0.6546 mL | 1.3093 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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