Z-VAD-FMK |
Catalog No.: GC12861 |
Z-VAD-FMK (Z-Val-Ala-Asp(OMe)-FMK) is a cell-permeable and irreversible pan-caspase inhibitor. Z-VAD-FMK is an ubiquitin carboxy-terminal hydrolase L1 (UCHL1) inhibitor. Z-VAD-FMK irreversibly modifies UCHL1 by targeting the active site of UCHL1.
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
- View current batch:
-
Purity: >99.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Cell experiment [1]: | |
Cell lines |
CD4+ and CD8+ T cells |
Preparation Method |
Soluble in DMSO to 20 mM |
Reaction Conditions |
100 μM, 24 h |
Applications |
Z-VAD-FMK is immunosuppressive in vitro and inhibit T cell proliferation without blocking the processing of caspase-8 and caspase-3. |
Animal experiment [2]: | |
Animal models |
C57BL/6 Mice (Treatment with LPS) |
Preparation Method |
Soluble in DMSO to 20 mM |
Dosage form |
20 μg/g, i.p. |
Applications |
Z-VAD-FMK treatment alleviates LPS-induced endotoxic shock by inducing macrophage necroptosis and promoting MDSC-mediated inhibition of macrophage activation. |
References: [1]. Lawrence CP, Chow SC. Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties. Toxicol Appl Pharmacol. 2012 Nov 15;265(1):103-12. [2]. Li X, Yao X, Zhu Y, et al. The Caspase Inhibitor Z-VAD-FMK Alleviates Endotoxic Shock via Inducing Macrophages Necroptosis and Promoting MDSCs-Mediated Inhibition of Macrophages Activation. Front Immunol. 2019 Aug 2;10:1824. |
Z-VAD-FMK (Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone), an ICE-like protease inhibitor, inhibits apoptosis by preventing the processing of CPP32 to its active form. [3]
Z-VAD-FMK is immunosuppressive in vitro and inhibit T cell proliferation without blocking the processing of caspase-8 and caspase-3. Z-VAD-FMK is capable of inhibiting T cell proliferation induced by anti-CD3 plus anti-CD28 or PHA. Besides, z-VAD-FMK inhibits caspase processing during apoptosis but not during T cell activation. Z-VAD-FMK Inhibits caspase processing and apoptosis induction in tumor cells in vitro (IC50 = 0.0015 - 5.8 mM). [1]
Z-VAD-FMK, can be used to induce necroptosis under certain stimuli. Treatment of mice with Z-VAD-FMK could significantly reduce mortality and alleviate disease after lipopolysaccharide (LPS) challenge. Notably, in LPS-challenged mice, treatment with Z-VAD-FMK could also reduce the percentage of peritoneal macrophages by promoting necroptosis and inhibiting pro-inflammatory responses in macrophages. What’s more, pretreatment with Z-VAD-FMK promoted LPS-induced nitric oxide-mediated necroptosis of bone marrow-derived macrophages (BMDMs), leading to reduced pro-inflammatory cytokine secretion. Interestingly, Z-VAD-FMK treatment promoted the accumulation of myeloid-derived suppressor cells (MDSCs) in a mouse model of endotoxin shock, and this process inhibited LPS-induced pro-inflammatory responses in macrophages. Treatment with Z-VAD-FMK alleviates LPS-induced endotoxic shock by inducing macrophage necroptosis and promoting MDSC-mediated inhibition of macrophage activation. For in vivo experienment, the mice were pretreated or post-treated with Z-VAD-FMK (5, 10, and 20 μg/g of body weight) or vehicle (saline) for 2 h and endotoxic shock was induced by an intraperitoneal injection of LPS (10 μg/g of body weight) and saline was used as control. [2]
References:
[1]. Lawrence CP, Chow SC. Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties. Toxicol Appl Pharmacol. 2012 Nov 15;265(1):103-12.
[2]. Li X, Yao X, Zhu Y, et al. The Caspase Inhibitor Z-VAD-FMK Alleviates Endotoxic Shock via Inducing Macrophages Necroptosis and Promoting MDSCs-Mediated Inhibition of Macrophages Activation. Front Immunol. 2019 Aug 2;10:1824.
[3]. Slee EA, Zhu H, et al. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32. Biochem J. 1996 Apr 1;315 (Pt 1) (Pt 1):21-4.
Cas No. | 187389-52-2 | SDF | |
Synonyms | Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone,Z-Val-Ala-Asp(OMe)-FMK | ||
Chemical Name | methyl (3S)-5-fluoro-3-[[(2S)-2-[[(2S)-3-methyl-2-(phenylmethoxycarbonylamino)butanoyl]amino]propanoyl]amino]-4-oxopentanoate | ||
Canonical SMILES | CC(C)C(C(=O)NC(C)C(=O)NC(CC(=O)OC)C(=O)CF)NC(=O)OCC1=CC=CC=C1 | ||
Formula | C22H30FN3O7 | M.Wt | 467.49 |
Solubility | ≥ 23.37mg/mL in DMSO | Storage | Store at -20°C |
General tips | For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months. | ||
Shipping Condition | Evaluation sample solution : ship with blue ice All other available size: ship with RT , or blue ice upon request |
Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)
Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)
Calculation results:
Working concentration: mg/ml;
Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid 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 master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL saline, mix and clarify.
Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.
Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such as vortex, ultrasound or hot water bath can be used to aid dissolving.
3. All of the above co-solvents are available for purchase on the GlpBio website.
Effect of caspase inhibitor Z-VAD-FMK on bovine sperm cryotolerance
The aim of this study was to evaluate the treatment of bovine semen with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), before or after freezing on semen quality. After the initial assessment, sperm from 4 bulls were pooled (Experiment 1) and cryopreserved in BioXcell containing 0, 20 and 100 μM Z-VAD-FMK. After thawing semen viability, motility, membrane integrity, as well as DNA fragmentation and ΔΨm were evaluated. In Experiment 2, bovine frozen/thawed sperm were incubated for 1 hr with 0, 20 and 100 ?M Z-VAD-FMK before assessing the semen quality. The treatment with Z -VAD-FMK before cryopreservation improved post-thawing sperm motility compared to the control group (p < .05), while no differences were recorded in sperm viability and membrane integrity among groups (on average 86.8 ± 1.5 and 69.1 ± 1.4, respectively). Interestingly, at the highest concentration, DNA fragmentation decreased (p < .05), while the percentage of spermatozoa with high ΔΨm increased (p < .05). The results of Experiment 2 showed that 1-hr treatment with Z-VAD-FMK did not affect sperm motility and viability (on average 63.4 ± 5.8 and 83.7.1 ± 1.2, respectively). However, Z-VAD-FMK improved sperm membrane integrity (p < .05) and at the highest concentration tested decreased the proportion of sperm showing DNA fragmentation (p < .05). No differences were recorded in the percentage of spermatozoa with high ΔΨm (on average 57.0 ± 11.4). In conclusion, the treatment with 100 ?M of the caspase inhibitor Z-VAD-FMK before freezing increased bovine sperm mass motility and ΔΨm, while decreasing sperm DNA fragmentation. Treatment of semen after thawing with 100 ?M Z-VAD-FMK improved sperm membrane integrity and reduced DNA fragmentation.
Effect of anti-apoptotic drug Z-VAD-FMK on in vitro viability of dog follicles
It is recognized that ovarian follicular atresia is associated with apoptosis, and the most important effector of cell death is caspase-3. The aim of this study was to investigate the influence of anti-apoptotic drug Z-VAD-FMK on in vitro follicle growth in the domestic dog. Ovaries were obtained from peri-pubertal and adult domestic dogs, and cortical fragments recovered and incubated on 1.5% (w/v) agarose gel blocks within a 24-well culture plate containing Minimum Essential Medium Eagle-Alpha Modification (αMEM) supplemented with 4.2 μg/mL insulin, 3.8 μg/mL transferrin, 5 ng/mL selenium, 2 mM L-glutamine, 100 μg/mL of penicillin G sodium, 100 μg/mL of streptomycin sulfate, 0.05 mM ascorbic acid, 10 ng/mL of FSH and 0.1% (w/v) polyvinyl alcohol in humidified atmosphere of 5% CO2 and 5% O2. The cortices were randomly allocated in six treatments: 1) 10 ng/mL EGF (EGF V0); 2) 10 ng/mL of EGF plus 1 mM Z-VAD-FMK (EGF V1); 3) 10 ng/mL of EGF and 10 mM Z-VAD-FMK (EGF V10); 4) 1 mM Z-VAD-FMK; 5) 10 mM Z-VAD-FMK and (6) no EGF and Z-VAD-FMK supplementation (Control). The cortices were processed for histology and assessed for viability (based on morphology), density of structurally normal follicles, and diameter immediately after collection (non-culture Control) or after 3 or 7 days of in vitro incubation. Evaluation of mRNA expression of Cas3 in fresh cortices and those incubated for 3 days was performed using real-time PCR. Histological analysis revealed that in vitro incubation decreased (P < 0.05) follicle viability and density compared to the fresh, non-culture control. Addition of 10 μM of Z-VAD-FMK alone to the culture medium sustained follicle viability at Day 3, but did not impact follicle diameter when compared to the other treatment groups (p < 0.001); however, the beneficial benefit of this anti-apoptotic drug diminished after 7 days of incubation. Furthermore, Z-VAD-FMK supplementation did not impact Cas3 expression. The findings demonstrated that dog ovarian tissues are highly susceptible to in vitro incubation and Z-VAD-FMK supported short-term survival of dog follicles enclosed within the ovarian cortex.
Role of HMGB1 in TNF-α Combined with Z-VAD-fmk-Induced L929 Cells Necroptosis
The present study established a necroptosis model in vitro and investigated the role of HMGB1 in cell necroptosis. A combination of tumor necrosis factor-α and z-VAD-fmk was used to induce necroptosis in L929 cells with necroptosis inhibitor necrostatin-1 applied as an intervention. Flow cytometry and transmission electron microscopy (TEM) were used to measure cell necroptosis. Western blotting assay was applied to detect the expression of receptor-interacting serine/threonine-protein kinase 3 (RIPK3), mixed lineage kinase domain-like pseudokinase (MLKL) and HMGB1. Co-immunoprecipitation (Co-IP) assay was used to confirm the interaction between HMGB1 and RIPK3. Our study demonstrated that HMGB1 migrated from the nucleus to the cytoplasm at the onset of necroptosis and was subsequently released passively to the extracellular matrix. Further experiments determined that the binding of HMGB1 with RIPK3 in the cytoplasm was loose during necroptosis. By contrast, when necroptosis was inhibited, the interaction in the cytoplasm was tight suggesting that this association between HMGB1 and RIPK3 might affect its occurrence. In conclusion, the transfer of HMGB1 from nucleus to cytoplasm, and its interaction with RIPK3 might be potentially involved in necroptosis.
Evaluation of Z-VAD-FMK as an anti-apoptotic drug to prevent granulosa cell apoptosis and follicular death after human ovarian tissue transplantation
Purpose: To evaluate the efficiency of ovarian tissue treatment with Z-VAD-FMK, a broad-spectrum caspase inhibitor, to prevent follicle loss induced by ischemia/reperfusion injury after transplantation.
Methods: In vitro, granulosa cells were exposed to hypoxic conditions, reproducing early ischemia after ovarian tissue transplantation, and treated with Z-VAD-FMK (50 μM). In vivo, cryopreserved human ovarian fragments (n = 39) were embedded in a collagen matrix containing or not Z-VAD-FMK (50 μM) and xenotransplanted on SCID mice ovaries for 3 days or 3 weeks.
Results: In vitro, Z-VAD-FMK maintained the metabolic activity of granulosa cells, reduced HGL5 cell death, and decreased PARP cleavage. In vivo, no improvement of follicular pool and global tissue preservation was observed with Z-VAD-FMK in ovarian tissue recovered 3-days post-grafting. Conversely, after 3 weeks of transplantation, the primary follicular density was higher in fragments treated with Z-VAD-FMK. This improvement was associated with a decreased percentage of apoptosis in the tissue.
Conclusions: In situ administration of Z-VAD-FMK slightly improves primary follicular preservation and reduces global apoptosis after 3 weeks of transplantation. Data presented herein will help to guide further researches towards a combined approach targeting multiple cell death pathways, angiogenesis stimulation, and follicular recruitment inhibition.
Caspase inhibitor z-VAD-FMK increases the survival of hair cells after Actinomycin-D-induced damage in vitro
Actinomycin-D (Act-D) is a highly effective chemotherapeutic agent that induces apoptosis in systemic tissues. Act-D combined with other chemotherapeutic agents exhibits ototoxic effects and causes hearing impairment. To investigate the potential toxic effects of Act-D in the inner ear, we treated cochlear organotypic cultures with varying concentrations of Act-D for different durations. For the first time, we found that Act-D specifically induced HC loss and apoptosis in a dose- and time-dependent manner but not neuronal degeneration. Co-treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK), a pan cysteine protease inhibitor, significantly reduced HC loss and apoptosis induced by Act-D, indicating increased cell survival. Taken together, Act-D exposure has ototoxic effects on the auditory system, while z-VAD-FMK prevents Act-D-induced hair cell damage.
Average Rating: 5
(Based on Reviews and 34 reference(s) in Google Scholar.)GLPBIO products are for RESEARCH USE ONLY. Please make sure your review or question is research based.
Required fields are marked with *