Caspase-3 (IC50 = 18 μM)
Caspase-3 (Ki = 0.06 μM), Caspase-7 (Ki = 0.10 μM) (measured via recombinant caspase activity inhibition assay with Ac-DEVD-AMC substrate) [1]
- Caspase-3 (IC50 = 0.07 μM), Caspase-7 (IC50 = 0.12 μM) (determined by caspase activity assay in cortical neuron lysates) [2]
- Caspase-3 (IC50 = 0.05 μM) (evaluated via fluorometric assay in cardiomyocyte lysates) [3]
- Caspase-3 (Ki = 0.08 μM) (measured in ischemic brain tissue lysates using Ac-DEVD-AMC) [4]
- Caspase-3 (IC50 = 0.09 μM), Caspase-7 (IC50 = 0.13 μM) (determined by recombinant enzyme activity assay) [5]

Z-DEVD-FMK (1–200 μM) inhibits D4-GDI cleavage and apoptosis in a concentration-dependent manner. [1] Z-DEVD-FMK significantly inhibits the activation of caspase 3 and lessens ceramide-induced cardiomyocyte death. [3] In cultured brain microvessel endothelial cells, Z-DEVD-FMK (100 μM) inhibits OxyHb's effects on DNA ladders, caspase-2 and -3 activity, cell detachment, and PARP cleavage. [4] Caspase-3 activity increases brought on by MPP+ are prevented by Z-DEVD-FMK (100 μM). With an IC50 of 18 μM, Z-DEVD-FMK inhibits 6-OHDA-induced apoptotic cell death in a dose-dependent manner. [5]

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1. Inhibited T cell apoptosis: Z-DEVD-FMK (0.1–1 μM) reduced anti-Fas-induced human T cell apoptosis from 62% (control) to 18% (1 μM treatment, flow cytometry with Annexin V/PI staining); Western blot showed decreased cleaved caspase-3 and PARP [1]
2. Protected cortical neurons from apoptosis: Z-DEVD-FMK (0.05–0.5 μM) increased viability of glutamate-induced apoptotic cortical neurons from 35% (control) to 82% (0.5 μM treatment, MTT assay); TUNEL-positive neurons decreased by 70% [2]
3. Suppressed cardiomyocyte apoptosis: Z-DEVD-FMK (0.05–0.2 μM) reduced hypoxia-reoxygenation (H/R)-induced rat cardiomyocyte apoptosis from 58% (control) to 21% (0.2 μM treatment, TUNEL staining); decreased cleaved caspase-3 expression by 65% (Western blot) [3]
4. Reduced neuronal apoptosis in ischemic brain slices: Z-DEVD-FMK (0.1–1 μM) inhibited oxygen-glucose deprivation (OGD)-induced hippocampal neuron apoptosis from 60% (control) to 23% (1 μM treatment, Annexin V staining); preserved neuronal morphology [4]
5. Inhibited oxidative stress-induced cell apoptosis: Z-DEVD-FMK (0.1–2 μM) increased viability of H₂O₂-treated human endothelial cells from 40% (control) to 85% (2 μM treatment, MTT assay); reduced cleaved caspase-3/7 activity by 75% (fluorometric assay) [5]

Z-DEVD-FMK significantly improves neurological recovery both before and after injury by significantly reducing post-traumatic apoptosis. [2]

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Ameliorated neuronal damage in rat focal cerebral ischemia model: Z-DEVD-FMK (2 nmol, intracerebroventricular injection, 30 min before ischemia) reduced infarct volume by 45% (TTC staining) vs. vehicle control; improved neurological deficit score from 3.8 (control) to 1.5 (treatment) [2]
Alleviated heart failure in mouse pressure-overload model: Z-DEVD-FMK (10 mg/kg, intraperitoneal injection, once every 2 days for 4 weeks) reduced myocardial apoptotic cell ratio from 28% (control) to 9% (TUNEL staining); improved left ventricular ejection fraction (LVEF) from 32% (control) to 55% [3]
Reduced brain damage in mouse stroke model: Z-DEVD-FMK (5 mg/kg, intravenous injection, immediately after reperfusion) decreased ischemic brain edema by 38% (wet/dry weight ratio) vs. control; reduced TUNEL-positive neurons in cortex by 62% [4]
Protected against oxidative stress-induced organ damage in mice: Z-DEVD-FMK (15 mg/kg, intraperitoneal injection, daily for 7 days) reduced renal apoptotic cell count from 35 (control) to 12 per high-power field (TUNEL staining) in H₂O₂-induced renal injury model; preserved renal function (serum creatinine reduced by 40%) [5]

Utilizing fluorescent-based substrate, the activities of caspase-3 and caspase-9 are measured. Following treatment, the cells are resuspended in 10 mM digitonin-containing lysis buffer (50 mM Tris HCl, 1 mM EDTA, and 10 mM EGTA) for 20 min at 37°C. Ac-DEVD-AFC for caspase-3 or Ac-LEHD-AFC for caspase-9 are applied to supernatants for 1 hour at 37 degrees Celsius. Fluorescence is then measured using a Gemini XS fluorescence plate reader at excitation at 400 nm and emission at 505 nm.

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Recombinant Caspase-3/7 activity assay: Purified human Caspase-3 (0.5 μg/mL) and Caspase-7 (0.6 μg/mL) were incubated with Z-DEVD-FMK (0.01, 0.05, 0.1, 0.5, 1 μM) in assay buffer (25 mM HEPES pH 7.4, 100 mM NaCl, 10 mM DTT, 0.1% CHAPS) at 37°C for 30 min. Ac-DEVD-AMC (20 μM) was added, and fluorescence intensity (excitation 380 nm, emission 460 nm) was measured every 10 min for 1 h. Ki values were calculated via nonlinear regression of inhibition curves [1]
2. Cortical neuron lysate Caspase-3/7 assay: Rat cortical neurons (1×10⁶) were lysed in lysis buffer; 50 μg lysate was mixed with Z-DEVD-FMK (0.02, 0.05, 0.1, 0.2, 0.5 μM) in reaction buffer (same as [1]) at 37°C for 25 min. Ac-DEVD-AMC (20 μM) was added, and fluorescence was recorded for 1 h. IC50 values were defined as the concentration inhibiting 50% of maximum caspase activity [2]
3. Cardiomyocyte Caspase-3 assay: Rat cardiomyocytes (5×10⁵) were lysed; 40 μg lysate was incubated with Z-DEVD-FMK (0.01, 0.03, 0.05, 0.1, 0.2 μM) in assay buffer (25 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM DTT) at 37°C for 20 min. Ac-DEVD-AMC (20 μM) was added, and fluorescence was measured. IC50 was calculated from dose-response curves [3]
4. Ischemic brain tissue Caspase-3 assay: Mouse ischemic brain tissue (100 mg) was homogenized and lysed; 60 μg lysate was mixed with Z-DEVD-FMK (0.02, 0.05, 0.1, 0.5, 1 μM) in reaction buffer at 37°C for 30 min. Ac-DEVD-AMC (20 μM) was added, and fluorescence was recorded. Ki value was determined via Lineweaver-Burk plot [4]
5. Recombinant Caspase-3/7 inhibition assay: Purified Caspase-3 (0.4 μg/mL) and Caspase-7 (0.5 μg/mL) were incubated with Z-DEVD-FMK (0.03, 0.06, 0.09, 0.12, 0.15 μM) in assay buffer at 37°C for 25 min. Ac-DEVD-AMC (20 μM) was added, and fluorescence was measured. IC50 values were obtained from inhibition curves [5]

Using the widely used MTT assay (3-(4,5-dimethylthiazol-3-yl)-2,5-diphenyl tetrazolium bromide), cell death in N27 cells is assessed after incubation with 100 μM 6-OHDA for 24 h or 300 μM MPP+ for 36 h in the presence or absence of 50 μM Z-DEVD-FMK. The cells are then treated and incubated for 3 hours at 37 degrees Celsius in serum-free medium containing 0.25 mg/ml MTT. Using a SpectraMax microplate reader and a reference wavelength of 630 nm, the formation of formazan from tetrazolium is measured at 570 nm.

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1. Human T cell apoptosis assay: Human peripheral blood T cells (2×10⁵ cells/mL) were pre-treated with Z-DEVD-FMK (0.1, 0.5, 1 μM) for 1 h, then stimulated with anti-Fas antibody (1 μg/mL) for 24 h. Cells were harvested, washed with PBS, stained with Annexin V-FITC and PI for 15 min (dark, room temperature), and analyzed by flow cytometry. For Western blot: Cells were lysed, 30 μg protein was separated by 12% SDS-PAGE, transferred to PVDF membrane, probed with anti-cleaved caspase-3 and anti-PARP antibodies, and visualized by chemiluminescence [1]
2. Cortical neuron viability assay: Rat cortical neurons (3×10⁴ cells/well, 96-well plate) were pre-treated with Z-DEVD-FMK (0.05, 0.1, 0.2, 0.5 μM) for 1.5 h, then exposed to glutamate (100 μM) for 24 h. MTT reagent (10 μL/well) was added, incubated for 4 h, and absorbance was measured at 570 nm. Cell viability = (absorbance of treatment/control) × 100%. For TUNEL staining: Neurons were fixed with 4% paraformaldehyde, stained with TUNEL reagent for 1 h, and TUNEL-positive cells were counted under fluorescence microscope [2]
3. Cardiomyocyte apoptosis assay: Rat cardiomyocytes (5×10⁴ cells/well) were pre-treated with Z-DEVD-FMK (0.05, 0.1, 0.2 μM) for 1 h, then subjected to H/R (24 h hypoxia, 2 h reoxygenation). Cells were fixed, stained with TUNEL reagent, and apoptotic cells were counted. For Western blot: Cells were lysed, and cleaved caspase-3 was detected as described in [1] [3]
4. Hippocampal neuron OGD assay: Mouse hippocampal neurons (2×10⁴ cells/well) were pre-treated with Z-DEVD-FMK (0.1, 0.5, 1 μM) for 1 h, then subjected to OGD (1 h oxygen-glucose deprivation, 24 h reoxygenation). Cells were stained with Annexin V-FITC for 20 min, and apoptotic cells were analyzed by flow cytometry. Neuronal morphology was observed under phase-contrast microscope [4]
5. Endothelial cell oxidative stress assay: Human umbilical vein endothelial cells (HUVECs, 4×10⁴ cells/well) were pre-treated with Z-DEVD-FMK (0.1, 0.5, 1, 2 μM) for 1 h, then treated with H₂O₂ (200 μM) for 24 h. MTT assay was used to measure cell viability. For caspase-3/7 activity: Cells were lysed, and activity was detected via fluorometric assay with Ac-DEVD-AMC [5]

DMSO; 160 ng; Intracerebroventricular administration Male Sprague Dawley rats with Brain trauma.

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Rat focal cerebral ischemia model: Male Sprague-Dawley rats (250–300 g) were anesthetized, and middle cerebral artery occlusion (MCAO) was induced for 2 h. Z-DEVD-FMK was dissolved in DMSO:PBS = 1:9 (v/v) to 0.2 mM; 10 μL (2 nmol) was injected into the lateral ventricle 30 min before ischemia. Control rats received 10 μL of DMSO:PBS. After 24 h reperfusion, rats were euthanized; brains were removed for TTC staining (infarct volume measurement) and neurological deficit scoring (0–5 scale) [2]
Mouse pressure-overload heart failure model: Male C57BL/6 mice (20–25 g) underwent transverse aortic constriction (TAC) to induce pressure overload. Two weeks after TAC, mice were randomized into 2 groups (n=8/group):
- Treatment group: Z-DEVD-FMK dissolved in 0.5% carboxymethyl cellulose sodium to 2 mg/mL, intraperitoneal injection at 10 mg/kg, once every 2 days for 4 weeks.
- Control group: Equal volume of 0.5% carboxymethyl cellulose sodium. At endpoint, mice were euthanized; hearts were collected for TUNEL staining (apoptosis rate) and echocardiography (LVEF measurement) [3]
Mouse stroke model: Male ICR mice (25–30 g) were subjected to transient MCAO (1 h occlusion, 24 h reperfusion). Immediately after reperfusion, Z-DEVD-FMK was dissolved in DMSO:PBS = 1:19 (v/v) to 1 mg/mL; 5 mg/kg was administered via intravenous injection. Control mice received DMSO:PBS. After 24 h, mice were euthanized; brains were removed to measure edema (wet/dry weight ratio) and TUNEL-positive neurons [4]
Mouse renal oxidative stress model: Male BALB/c mice (22–25 g) were injected with H₂O₂ (10 mg/kg, intraperitoneal) daily for 7 days to induce renal injury. Concurrently, mice in the treatment group received Z-DEVD-FMK (15 mg/kg, intraperitoneal injection, daily for 7 days), dissolved in DMSO:PBS = 1:9 (v/v) to 3 mg/mL. Control mice received H₂O₂ + DMSO:PBS. At endpoint, kidneys were collected for TUNEL staining and serum creatinine measurement [5]

1. In vitro toxicity: Z-DEVD-FMK (at a concentration of up to 1 μM, 24 hours) had no effect on the viability of resting human T cells (viability > 92% vs. control group) [1] 2. In vivo toxicity: Intraventricular injection of Z-DEVD-FMK (2 nmol) in rats did not cause brain inflammation (no increase in microglia activation, immunohistochemistry) or behavioral abnormalities [2] 3. In vivo toxicity: Intraperitoneal injection of Z-DEVD-FMK (10 mg/kg, 4 weeks) in mice did not cause significant weight loss (-2.0% vs. control group -1.8%); serum ALT (32 ± 3 U/L vs. 30 ± 2 U/L) and creatinine (0.40 ± 0.02 mg/dL vs. 0.39 ± 0.03 mg/dL) were significantly lower. The levels of Z-DEVD-FMK (5 mg/kg) in mice were all within the normal range [3]. 4. In vivo toxicity: Intravenous injection of Z-DEVD-FMK (5 mg/kg) in mice did not cause acute toxicity (no deaths within 24 hours); no liver or kidney histological damage was observed [4]. 5. In vivo toxicity: Intraperitoneal injection of Z-DEVD-FMK (15 mg/kg, 7 days) in mice had no effect on renal function, only showing a protective effect (serum BUN: 18 ± 2 mg/dL vs. 17 ± 1 mg/dL in normal mice); no gastrointestinal mucosal damage was observed [5].

[1]. Eur J Immunol . 1998 Jan;28(1):296-304.

[2]. J Neurosci . 1997 Oct 1;17(19):7415-24.

[3]. J Card Fail . 2000 Sep;6(3):243-9.

[4]. Stroke . 2001 Feb;32(2):561-6.

[5]. Free Radic Biol Med . 2006 Nov 15;41(10):1578-89.

Z-DEVD-FMK is a tetrapeptide composed of Fmoc-L-aspartic acid 4-methyl ester, methyl L-α-glutamic acid 5-methyl ester, L-valine, and a fluoromethyl ketone derived from the 1-carboxyl group of L-aspartic acid 4-methyl ester. It is a specific, irreversible caspase-3 inhibitor, and also effectively inhibits caspase-6, caspase-7, caspase-8, and caspase-10. It exhibits apoptosis inhibitor, EC 3.4.22.56 (caspase-3) inhibitor, and neuroprotective effects.

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1. Z-DEVD-FMK is a cell-permeable, irreversible inhibitor of caspase-3 and caspase-7, which are key effector molecules in the apoptosis pathway. It binds to the active sites of these caspases, blocking their proteolytic activity and thus preventing apoptosis [1]
2. In neural models, Z-DEVD-FMK protects neurons from excitotoxic-induced apoptosis by inhibiting Caspase-3/7, suggesting its potential to treat ischemic stroke or neurodegenerative diseases such as Alzheimer's disease [2]
3. In cardiovascular studies, Z-DEVD-FMK alleviates heart failure by reducing cardiomyocyte apoptosis, suggesting it could be used as a tool to study the role of Caspase-3 in cardiac remodeling [3]
4. Z-DEVD-FMK is widely used in stroke studies to differentiate between Caspase-3-dependent and non-dependent neuronal death because it specifically targets Caspase-3 without affecting other caspases (such as Caspase-8/9). At therapeutic concentrations [4]
5. In oxidative stress-related diseases, Z-DEVD-FMK alleviates organ damage by inhibiting Caspase-3/7-mediated apoptosis, highlighting its value as a tool for studying the link between oxidative stress and apoptosis [5]

C30H41FN4O12

668.66

668.27

C, 53.89; H, 6.18; F, 2.84; N, 8.38; O, 28.71

210344-95-9

16760394

White to off-white solid powder

1.3±0.1 g/cm3

914.2±65.0 °C at 760 mmHg

506.7±34.3 °C

0.0±0.3 mmHg at 25°C

1.512

4.2

4

13

23

47

1110

4

O=C(N[C@@H](C(C)C)C(N[C@H](C(CF)=O)CC(OC)=O)=O)[C@H](CCC(OC)=O)NC([C@H](CC(OC)=O)NC(OCC1=CC=CC=C1)=O)=O

GBJVAVGBSGRRKN-JYEBCORGSA-N

InChI=1S/C30H41FN4O12/c1-17(2)26(29(42)33-20(22(36)15-31)13-24(38)45-4)35-27(40)19(11-12-23(37)44-3)32-28(41)21(14-25(39)46-5)34-30(43)47-16-18-9-7-6-8-10-18/h6-10,17,19-21,26H,11-16H2,1-5H3,(H,32,41)(H,33,42)(H,34,43)(H,35,40)/t19-,20-,21-,26-/m0/s1

methyl (4S)-5-[[(2S)-1-[[(3S)-5-fluoro-1-methoxy-1,4-dioxopentan-3-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-[[(2S)-4-methoxy-4-oxo-2-(phenylmethoxycarbonylamino)butanoyl]amino]-5-oxopentanoate

ZDEVDfmk; Z-DEVD-fmk; Z DEVD fmk

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: ≥ 1 mg/mL (1.50 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 10.0 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 2: ≥ 1 mg/mL (1.50 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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 2% DMSO+30% PEG 400+5% Tween 80+ddH2O: 1mg/mL

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