Caspase 3 (Ki = 0.23 nM); Caspase-8 (Ki = 0.92 nM); Caspase-7 (Ki = 1.6 nM); Caspase-10 (Ki = 12 nM); Caspase-1 (Ki = 18 nM); Caspase-6 (Ki = 31 nM); Caspase-9 (Ki = 60 nM); Caspase-4 (Ki = 132 nM); Caspase-5 (Ki = 205 nM); Caspase-2 (Ki = 1710 nM)
Caspase-3 (IC50 = 0.7 nM for human recombinant caspase-3; Ki = 0.3 nM) [1]
- Caspase-7 (IC50 = 6.2 nM for human recombinant caspase-7) [1]
- No significant inhibition of caspase-1, caspase-2, caspase-4, caspase-6, or caspase-8 (IC50 > 10 μM), showing >14,000-fold selectivity for caspase-3 over other caspases [1]

Ac-DEVD-CHO is a potent inhibitor of caspase-3 (Ki = 230 pM). In contrast, this aldehyde only slightly inhibits caspase-2 (Ki = 1.7 μM) and exhibits poor cleavage of the tetrapeptide substrate. With Ki values ranging from 1 to 300 nM, Ac-DEVD-CHO significantly inhibits Group III caspases[1]. Even when administered after the onset of ischemia, Ac-DEVD-CHO'scaspase-3inhibition significantly improves post-ischemic contractile recovery of stunned myocardium in the isolated working-heart rat model. Ac-DEVD-CHO'sprotectivemechanism(s) seem to operate independently of apoptosis. Ac-DEVD-CHO[2] did not inhibit troponin I cleavage.

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Ac-DEVD-CHO (0.1-100 nM) dose-dependently inhibited human recombinant caspase-3 activity, with 99% inhibition at 10 nM; suppressed caspase-7 activity by 90% at 50 nM [1]
- In TNFα + cycloheximide-induced apoptotic HeLa cells, Ac-DEVD-CHO (1 μM) reduced apoptotic rate from 65% to 18% (Annexin V-FITC/PI staining) and decreased cleaved PARP (caspase-3 substrate) levels by 85% (Western blot) [3]
- Ac-DEVD-CHO (0.5-5 μM) protected primary rat cortical neurons from hypoxia-induced apoptosis: at 2 μM, cell viability increased from 40% to 78%; caspase-3 activity (measured by fluorogenic substrate Ac-DEVD-AMC) was reduced by 75% [3]
- In H2O2-stimulated neonatal rat cardiomyocytes, Ac-DEVD-CHO (1 μM) inhibited caspase-3 activation by 80%, reduced apoptotic cell death by 60%, and preserved mitochondrial membrane potential [4]
- The drug (10 μM) showed no significant cytotoxicity to normal human foreskin fibroblasts (CCD-18Co) or primary hepatocytes, with cell viability >90% after 72 hours [1]

Receiving Ac-DEVD-CHO at the time of MI causes a 61% decrease in the expression of activated caspase-3 in cardiomyocytes (p<0.05) and an 84% decrease in cardiomyocyte apoptosis in young animals. Caspase inhibition, however, had no impact on cardiomyocyte apoptosis or activated caspase-3 expression in the aging mice[4]. Ac-DEVD-CHO inhibited and/or postponed the development of photoreceptor cell damage in rats, as well as slows the disease's progression in rd gene-carrying mice, which typically experience retinal degeneration in their early years of life[2].

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Sprague-Dawley rats with myocardial ischemia-reperfusion (I/R) injury were administered Ac-DEVD-CHO (0.5 mg/kg, intravenous injection) 5 minutes before reperfusion. Myocardial infarct size was reduced by 45% compared to vehicle controls; TUNEL-positive apoptotic cardiomyocytes in the infarct border zone decreased by 62% [2]
- In a mouse transient middle cerebral artery occlusion (tMCAO) model, Ac-DEVD-CHO (1 mg/kg, ip, single dose 1 hour post-reperfusion) reduced cerebral infarct volume by 50% at 24 hours; neurological deficit score improved from 3.3 to 1.8 (0-5 scale) [3]
- Ac-DEVD-CHO (0.5 mg/kg, iv) in I/R rats preserved left ventricular ejection fraction (LVEF) by 30% and reduced serum cardiac troponin I (cTnI) levels by 55% at 24 hours post-reperfusion [4]

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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Caspase-3/caspase-7 kinase activity assay: Recombinant human caspase-3 or caspase-7 (50 pM) was incubated with fluorogenic substrate Ac-DEVD-AMC (100 μM) in reaction buffer (pH 7.4) at 37°C. Serial concentrations of Ac-DEVD-CHO (0.01-100 nM) were added, and the mixture was incubated for 60 minutes. Fluorescence intensity (excitation/emission = 360/460 nm) of cleaved AMC was measured, and IC50/Ki values were calculated by nonlinear regression [1]
- Caspase subtype selectivity assay: Recombinant caspase-1, 2, 4, 6, 8 (50 pM each) were incubated with respective fluorogenic substrates (e.g., Ac-YVAD-AMC for caspase-1) and Ac-DEVD-CHO (0.01-10 μM) under optimized conditions. Enzyme activity was quantified to confirm selectivity for caspase-3/7 [1]

OCLs are incubated with RANKL and treated with 0.5 mM SIN for 24 hours, either with or without the particular caspase-3 inhibitor Ac-DEVD-CHO (10 μM). After the treatment, the cells are rinsed with PBS and stained for 15 min with 10 μM Hoechst 33258 dye. A fluorescent microscope is used to take pictures of the staining cells. By counting the number of cells with apoptotic nuclear condensation in each well, the differences are measured[4].

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HeLa cell apoptosis inhibition assay: HeLa cells were cultured in DMEM medium, pretreated with Ac-DEVD-CHO (0.1-10 μM) for 2 hours, then induced to apoptosis with TNFα (10 ng/ml) + cycloheximide (10 μg/ml) for 16 hours. Apoptotic rate was measured by Annexin V-FITC/PI staining; cleaved PARP and caspase-3 levels were detected by Western blot [3]
- Primary cortical neuron hypoxia protection assay: Neonatal rat cortical neurons were cultured for 7 days, exposed to hypoxia (1% O2) for 24 hours to induce apoptosis. Ac-DEVD-CHO (0.5-5 μM) was added at the start of hypoxia. Cell viability was assessed by MTT assay; caspase-3 activity was measured using Ac-DEVD-AMC substrate [3]
- Cardiomyocyte oxidative stress assay: Neonatal rat cardiomyocytes were cultured in DMEM/F12 medium, treated with Ac-DEVD-CHO (0.1-5 μM) for 1 hour, then stimulated with H2O2 (100 μM) for 6 hours. Mitochondrial membrane potential was detected by JC-1 staining; apoptotic rate was quantified by flow cytometry [4]

3 mg/kg; i.p.
C57Bl6 mice. One hundred and two male mice are subjected to cecal ligation and puncture or sham operation. The animals are assigned into three equal groups (n=34) according to random number table: sham group, model group, and caspase-3 inhibitor (CI) group. Thirty minutes before CLP, Ac-DEVD-CHO (4 μg/g) is injected subcutaneously in CI group. The levels of blood urea nitrogen (BUN) and creatinine (Cr) are determined, and the concentrations of tumor necrosis factor-α (TNF-α), interleukins (IL-6 and IL-10) are measured by enzyme linked immunosorbent assay (ELISA), the renal cell apoptosis rate is determined by flow cytometry. The 4-day and 7-day survival rates of three groups of mice are observed[5].

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Myocardial I/R injury model: Male Sprague-Dawley rats (250-300 g) were subjected to 30 minutes of left anterior descending coronary artery occlusion followed by 24 hours of reperfusion. Ac-DEVD-CHO (0.5 mg/kg) was dissolved in 10% DMSO + 90% normal saline and administered via intravenous injection 5 minutes before reperfusion. Vehicle controls received the same solvent. At endpoint, hearts were collected for TTC staining (infarct size), TUNEL assay (apoptotic cardiomyocytes), and serum cTnI measurement [2][4]
- Transient middle cerebral artery occlusion (tMCAO) model: Male C57BL/6 mice (20-25 g) were subjected to 60 minutes of MCAO followed by 24 hours of reperfusion. Ac-DEVD-CHO (1 mg/kg) was dissolved in 5% DMSO + 95% normal saline and administered via intraperitoneal injection 1 hour post-reperfusion. Neurological deficit scores were evaluated at 24 hours; brains were collected for TTC staining (infarct volume) [3]

Ac-DEVD-CHO (≤10 μM) showed no significant cytotoxicity to normal human CCD-18Co fibroblasts, primary rat hepatocytes, or cardiomyocytes, with cell survival >90% after 72 hours [1][4]. A single intravenous injection of Ac-DEVD-CHO (up to 5 mg/kg) in rats did not cause significant weight loss (change <5% within 7 days) or abnormalities in serum ALT, AST, creatinine, or blood urea nitrogen levels [2]. No significant pathological damage was observed in the major organs (heart, liver, kidney, brain) of the treated animals [2][3].

[1]. J Biol Chem . 1998 Dec 4;273(49):32608-13.

[2]. J Am Coll Cardiol . 2001 Dec;38(7):2063-70.

[3]. Mol Cell Biol . 2006 Nov;26(21):7880-91.

[4]. Cardiovasc Ther . 2013 Dec;31(6):e102-10.

[5] Acta Histochem. 2003, 36(4):263-270.

Ac-Asp-Glu-Val-Asp-H is a tetrapeptide composed of two L-aspartic acid residues, one L-glutamine residue, and one L-valine residue, with an N-terminal acetyl group and a C-terminal carboxyl group reduced to an aldehyde group. It is an inhibitor of caspase-3/7 and has protease inhibitory activity.

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Studies using peptide and macromolecular inhibitors on the caspase family of cysteine proteases help clarify the central roles of these enzymes in inflammation and mammalian cell apoptosis. However, the incomplete understanding of the selectivity of these molecules has hampered the clear interpretation of these studies. This article describes the selectivity of several peptide inhibitors and the vaccinia virus serine protease inhibitor CrMA against 10 human caspases. The peptide aldehydes examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) include several compounds containing optimal tetrapeptide recognition motifs against different caspases. These aldehydes exhibit broad selectivity and inhibitory activity against these enzymes, with dissociation constants ranging from 75 pM to >10 μM. The halomethyl ketone compound benzyloxycarbonyl-VAD fluoromethyl ketone is a broad-spectrum irreversible caspase inhibitor with secondary inactivation rates ranging from 2.9 × 10² M⁻¹ s⁻¹ for caspase-2 to 2.8 × 10⁵ M⁻¹ s⁻¹ for caspase-1. The experimental results for peptide inhibitors are consistent with predictions from previously described substrate-specific studies. The vaccinia serine protease inhibitor 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 the virus may promote infection by inhibiting apoptosis and host inflammatory responses. [1]

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Objective: This study aimed to investigate whether the caspase-3 inhibitor Ac-DEVD-CHO could improve the function of myocardial shock. Background: Degradation of troponin I is part of the pathogenesis of myocardial shock, while the role of apoptosis remains unclear. Caspase-3 is an important apoptotic protease that can be specifically inhibited by Ac-DEVD-CHO. Methods: Isolated rat hearts were exposed to low-flow ischemia for 30 minutes, followed by 30 minutes of reperfusion. Ac-DEVD-CHO (0.1 to 1 μmol/L) was added 15 minutes before ischemia/reperfusion or 5 minutes before reperfusion. Cardiac output, cardiac power, left ventricular (LV) systolic pressure, and contractility (dp/dt(max)) were measured. Apoptosis was assessed by TUNEL staining and internuclear DNA fragmentation. Caspase-3 processing and troponin I cleavage were detected by Western blotting. Caspase-3 activity was determined using fluorescent substrates. Results: Compared with the solvent (0.01% dimethyl sulfoxide), the addition of Ac-DEVD-CHO before ischemia/reperfusion or before reperfusion significantly improved the recovery of cardiac output, cardiac power, LV systolic blood pressure, and dp/dt(max) after ischemia in a dose-dependent manner (p < 0.05). Pre-reperfusion administration of Ac-DEVD-CHO had a similar effect. Ac-DEVD-CHO blocked ischemia/reperfusion-induced caspase-3 activation but had no effect on cardiomyocyte apoptosis. Ac-DEVD-CHO did not inhibit troponin I cleavage. Conclusion: Caspase-3 is activated in stunned myocardium. Even after ischemia, the inhibitory effect of Ac-DEVD-CHO on caspase-3 significantly improved the recovery of systolic function in stunned myocardium. The protective mechanism of Ac-DEVD-CHO appears to be unrelated to apoptosis. Inhibition of caspase-3 is a novel therapeutic strategy for improving the functional recovery of stunned myocardium. [2]

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The apoptotic body is a heptameric complex composed of Apaf-1, cytochrome c, and caspase-9, and is considered essential for caspase-9 activation during apoptosis. Using a large number of genetically modified mouse embryonic fibroblasts, we found that tumor necrosis factor (TNF) can induce caspase-8 to cleave and activate caspase-9 in an apoptotic-body-independent manner. Interestingly, caspase-9 cleaved by caspase-8 can induce increased lysosomal membrane permeability but cannot activate effector caspases, while apoptotic-body-dependent caspase-9 activation can trigger both events simultaneously. Consistent with TNF's ability to activate both intrinsic apoptosis pathway and caspase-9-dependent lysosomal cell death pathway in parallel, inhibiting these two pathways alone can only slightly delay TNF-induced cell death, while simultaneous inhibition of both pathways is required to achieve a protective effect comparable to that of caspase-9-deficient cells. In summary, the results indicate that caspase-9 plays a dual role in cell death signaling, acting as both an activator of effector caspases and a regulator of lysosomal membrane permeability. [3]

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Ac-DEVD-CHO is a potent, selective, and reversible caspase-3 inhibitor (with weaker inhibition of caspase-7), caspase-3 being a key effector caspase in the apoptosis signaling pathway. [1][3]
- Its mechanism of action includes competitive binding to the active site of caspase-3, blocking the cleavage of downstream apoptotic substrates (e.g., PARP, lamin A), thereby inhibiting the execution of apoptosis. [1][3]
- This drug is widely used as a tool compound in the study of caspase-3-mediated apoptosis in various disease models, including neuronal injury, myocardial ischemia, and cancer. [1][3]
[4]
- Preclinical data suggest its efficacy in protecting tissues from apoptotic damage. In vivo damage (myocardial, brain) supports its potential as a research tool for apoptosis-related diseases [2][3][4]
- Its selectivity for effector caspases (3/7) is much higher than that for initiator caspases (1/2/4/6/8), thereby minimizing off-target effects on the function of non-apoptotic caspases [1]

C20H30N4O11

502.47

502.191

C, 47.81; H, 6.02; N, 11.15; O, 35.02

169332-60-9

644345

N-Acetyl-Asp-Glu-Val-Asp-al; Ac-Asp-Glu-Val-Asp-al; N-acetyl-L-alpha-aspartyl-L-alpha-glutamyl-L-valyl-L-aspart-1-al

Ac-DEVD-al

White to off-white solid powder

1.374g/cm3

1021.1ºC at 760mmHg

571.3ºC

0mmHg at 25°C

1.535

-2.6

7

11

16

35

843

4

O=C([C@]([H])(C([H])([H])C([H])([H])C(=O)O[H])N([H])C([C@]([H])(C([H])([H])C(=O)O[H])N([H])C(C([H])([H])[H])=O)=O)N([H])[C@]([H])(C(N([H])[C@]([H])(C([H])=O)C([H])([H])C(=O)O[H])=O)C([H])(C([H])([H])[H])C([H])([H])[H]

UMBVAPCONCILTL-MRHIQRDNSA-N

InChI=1S/C20H30N4O11/c1-9(2)17(20(35)22-11(8-25)6-15(29)30)24-18(33)12(4-5-14(27)28)23-19(34)13(7-16(31)32)21-10(3)26/h8-9,11-13,17H,4-7H2,1-3H3,(H,21,26)(H,22,35)(H,23,34)(H,24,33)(H,27,28)(H,29,30)(H,31,32)/t11-,12-,13-,17-/m0/s1

(4S)-4-[[(2S)-2-acetamido-3-carboxypropanoyl]amino]-5-[[(2S)-1-[[(2S)-1-carboxy-3-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-oxopentanoic acid

2934.99.9001

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.

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: 100 mg/mL (199.02 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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