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Purity: ≥98%
Z-VAD-FMK (Caspase Inhibitor VI; Z-VAD(OH)-FMK) is a novel, potent and irreversible pan caspase inhibitor. For in vitro studies, there is no need for a pretreatment with esterase. Z-VAD(OH)-FMK, which is not methylated, is a form of Z-VAD-FMK that is helpful in studies involving recombinant or purified enzymes.
| Targets |
Caspase
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|---|---|
| ln Vitro |
Z-VAD(OH)-FMK, which is not methylated, is a form of Z-VAD-FMK that is helpful in studies involving recombinant or purified enzymes. [1]
Several natural products have been demonstrated to both enhance the anti-tumor efficacy and alleviate the side effects of conventional chemotherapy drugs. Rhein, a main constituent of the Chinese herb rhubarb, has been shown to induce apoptosis in various cancer types. However, the exact pharmacological mechanisms controlling the influence of Rhein on chemotherapy drug effects in pancreatic cancer (PC) remain largely undefined. In this study, we found that Rhein inhibited the growth and proliferation of PC cells through G1 phase cell cycle arrest. Moreover, Rhein induced caspase-dependent mitochondrial apoptosis of PC cells through inactivation of the PI3K/AKT pathway. Combination treatment of Rhein and oxaliplatin synergistically enhanced apoptosis of PC cells through increased generation of intracellular reactive oxygen species (ROS) and inactivation of the PI3K/AKT pathway. Pre-treatment with the ROS scavenger N-acetyl-L-cysteine attenuated the combined treatment-induced apoptosis and restored the level of phosphorylated AKT, indicating that ROS is an upstream regulator of the PI3K/AKT pathway. The combination therapy also exhibited stronger anti-tumor effects compared with single drug treatments in vivo. Taken together, these data demonstrate that Rhein can induce apoptosis and enhance the oxaliplatin sensitivity of PC cells, suggesting that Rhein may be an effective strategy to overcome drug resistance in the chemotherapeutic treatment of PC[2]. |
| ln Vivo |
Z-VAD-FMK, a widely used broad-spectrum caspase inhibitor, repairs muscle damage brought on by compression and preserves muscle function.
The purpose of the study was to evaluate the therapeutic benefit of treatments with carfilzomib (CFZ) and z-VAD-fmk in a mouse model of cancer-induced cachexia. The model of cancer-associated cachexia was generated by injecting murine C26 adenocarcinoma cells into BALB/C mice. CFZ and z-VAD-fmk were administered individually or in combination at 5 and 12 days after inoculation. Changes in body weight, gastrocnemius muscle mass, tumor burden, spontaneous activity, survival, and metabolic profiles were noted. Also evaluated were the circulatory levels of renin and angiotensin II, and levels of apoptotic, proteolytic, and renin-angiotensin system-associated markers and transcription factor 2 (ATF2) in gastrocnemius muscle. The CFZ and z-VAD-fmk treatments were associated with less muscle wasting, reduced tumor burden, modulated metabolism, higher levels of glucose, albumin, and total proteins, and lower levels of triglyceride fatty acids, more spontaneous physical activity, and longer survival in C26-inoculated mice compared with PBS-treated cachectic mice. CFZ and z-VAD-fmk treatments resulted in higher levels of caspase-3 and BAX and lower level of BCL-XL in gastrocnemius muscles and altered the level of proteins in the renin-angiotensin system. The combined treatment administered 5 days after C26 inoculation was more effective than other regimens. Combined treatment with CFZ and z-VAD-fmk early in the development of cachexia was associated with signs of less proteolysis and apoptosis and less severe cachexia in a mouse model of cancer-induced cachexia[3]. |
| Enzyme Assay |
Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes, with dissociation constants ranging from 75 pM to >10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki < 20 nM) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and most Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response[1].
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| Cell Assay |
Cell viability assay[2]
Cell viability was assessed with the Cell Counting Kit-8 (CCK-8) assay according to manufacturer's instructions. In brief, 3*103 cells in 100 μl culture medium were plated in a 96-well plate. After adherence, cells were treated with reagents for certain time as indicated by the figures. Subsequently, culture media were replaced and 10 μl CCK-8 solution were added to each well and incubated in 37 ℃ for 1 h. Absorbance at 450nm was measured using a microplate reader. To investigate the combined effect of oxaliplatin and Rhein, cells were treated with different ratio of drug concentrations and combination index (CI) was calculated using CalcuSyn software. Colony formation assay[2] To explore the long-term effects of drug treatment, 1*103 cells were seeded in 60 mm dish. After adherence, cells were treated with drugs for indicated concentrations for 24 h. Media were replaced by complete cell cultural medium without drug every 2-5 days. On day 14, cells were washed with PBS twice, fixed with 4% paraformaldehyde for 30 min and stained by 0.1% crystal violet for 30 min. Colonies were then photographed and counted. Cell cycle analysis and hypodiploid cell population determination[2] After treated with indicated drugs, cells were harvested and washed with PBS before fixing with cold ethanol (70% v/v) at 4℃ for 24 h. Cells were then washed, resuspended with cold PBS and 20 μl RNase A (50 μg/ml) were added and incubated at 37℃ for 30 min. 20 μl propidium iodide (PI) (50 μg/ml) were added and incubated in dark at 4℃ for 30 min. Distribution of cells with different DNA content or hypodiploid (sub-G1) cell populations which indicated apoptosis were then analyzed by flow cytometry on FACS Calibur flow cytometer. |
| Animal Protocol |
Male BALB/C mice
1.5 mg/kg s.c. To induce cancer cachexia, C26 cells growing in exponential phase were harvested with trypsin and injected subcutaneously into an axilla of the mouse. A total of 175 animals received C26 cell injections with 1 × 106 cells per site; 10 animals received PBS injection instead of C26 cells to serve as healthy controls. The tumor-bearing animals were divided into 7 groups of 25 animals each, according to the treatment and the time when the treatment started: CFZ (2 mg/kg, twice a week) and z-VAD-fmk (1.5 mg/kg, daily), alone (designated as “C” or “Z,” respectively) or in combination (designated as “U”). Each of these treatments was administered either 5 days after cell inoculation (preventive), when the tumor nodules were palpable, or 12 days after cell inoculation (post-cachexia), when the mice presented signs of cachexia. In addition, a group of tumor-bearing mice received sterile phosphate-buffered saline (PBS) to serve as the cachexia control (CC); another group of mice received subcutaneous injection of PBS, instead of C26, were the healthy controls (HC).[3] |
| References | |
| Additional Infomation |
Non-methylated, competitive, and irreversible inhibitor of caspase 1, as well as other caspases,1 which can be used directly with purified enzymes. It does not require an esterase to hydrolyze the O-methyl ester like the cell-permeable form, Z-Val-Ala-Asp(O-Me) fluoromethyl ketone.
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| Molecular Formula |
C21H28FN3O7
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|---|---|
| Molecular Weight |
453.46
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| Exact Mass |
453.191
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| Elemental Analysis |
C, 55.62; H, 6.22; F, 4.19; N, 9.27; O, 24.70
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| CAS # |
161401-82-7
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| Related CAS # |
Z-VAD(OMe)-FMK;187389-52-2
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| PubChem CID |
5497171
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| Appearance |
White to light yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
758.0±60.0 °C at 760 mmHg
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| Flash Point |
412.2±32.9 °C
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| Vapour Pressure |
0.0±2.7 mmHg at 25°C
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| Index of Refraction |
1.525
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| LogP |
3.04
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
13
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| Heavy Atom Count |
32
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| Complexity |
680
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| Defined Atom Stereocenter Count |
3
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| SMILES |
FCC([C@H](CC(=O)O)N([H])C([C@H](C)N([H])C([C@H](C(C)C)N([H])C(=O)OCC1C=CC=CC=1)=O)=O)=O
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| InChi Key |
SUUHZYLYARUNIA-YEWWUXTCSA-N
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| InChi Code |
InChI=1S/C21H28FN3O7/c1-12(2)18(25-21(31)32-11-14-7-5-4-6-8-14)20(30)23-13(3)19(29)24-15(9-17(27)28)16(26)10-22/h4-8,12-13,15,18H,9-11H2,1-3H3,(H,23,30)(H,24,29)(H,25,31)(H,27,28)/t13-,15-,18-/m0/s1
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| Chemical Name |
(3S)-5-fluoro-3-[[(2S)-2-[[(2S)-3-methyl-2-(phenylmethoxycarbonylamino)butanoyl]amino]propanoyl]amino]-4-oxopentanoic acid
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| Synonyms |
Z-Val-Ala-Asp-(OH)-Fluoromethyl Ketone; Z-VAD(OH)-FMK
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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 (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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: 90~100 mg/mL (198.5~220.5 mM)
Ethanol: ~90 mg/mL (~198.5 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.59 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 20.8 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: 2.08 mg/mL (4.59 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 20.8 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: ≥ 2.08 mg/mL (4.59 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 | 2.2053 mL | 11.0263 mL | 22.0527 mL | |
| 5 mM | 0.4411 mL | 2.2053 mL | 4.4105 mL | |
| 10 mM | 0.2205 mL | 1.1026 mL | 2.2053 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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