Caspase-1; IL-1β

Ac-YVAD-CHO acetate suppresses human and mouse IL-1β with IC50 values of 2.5 and 0.7 μM, respectively [1]. The rise in IL-1β in LPS-treated plasma and peritoneal fluid can be decreased by Ac-YVAD-CHO (0.01-100 μM) acetate [1]. Thymocyte apoptosis mediated by NO is decreased by Ac-YVAD-CHO (15.6 μM) acetate [3]. In thymocytes treated with SNAP, Ac-YVAD-CHO (15.6 μM, 12 hours) acetate prevents NO-induced PARP cleavage [3].

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The pan-caspase inhibitor, ZVAD-fmk, and the caspase-1 inhibitor, Ac-YVAD-CHO, both inhibited NO-induced thymocyte apoptosis in a dose-dependent manner, whereas the caspase-3 inhibitor, Ac-DEVD-cho, had little effect even at concentrations up to 500 microM. ZVAD-fmk and Ac-YVAD-CHO were able to inhibit apoptosis when added up to 12 h, but not 16 h, after treatment with the NO donor S-nitroso-N-acetyl penicillamine (SNAP). Caspase-1 activity was up-regulated at 4 h and 8 h and returned to baseline by 24 h; caspase-3 activity was not detected. Cytosolic fractions from SNAP-treated thymocytes cleaved the inhibitor of caspase-activated deoxyribonuclease. Such cleavage was completely blocked by Ac-YVAD-cho, but not by Ac-DEVD-cho or DEVD-fmk. Poly(ADP-ribose) polymerase (PARP) was also cleaved in thymocytes 8 h and 12 h after SNAP treatment; addition of Ac-YVAD-cho to the cultures blocked PARP cleavage. Furthermore, SNAP induced apoptosis in 44% of thymocytes from wild-type mice; thymocytes from caspase-1 knockout mice were more resistant to NO-induced apoptosis. These data suggest that NO induces apoptosis in thymocytes via a caspase-1-dependent but not caspase-3-dependent pathway. Caspase-1 alone can cleave inhibitor of caspase-activated deoxyribonuclease and lead to DNA fragmentation, thus providing a novel pathway for NO-induced thymocyte apoptosis[3].

Ac-YVAD-CHO (30 mg/kg; intraperitoneal; 6 hours) In the blood of mice sensitive to P. acnes, acetate lowers the levels of IL-1β [1]. Intrastriatal infusion of Ac-YVAD-CHO (2–8 μg) Rat striatal apoptosis induced by quinolinic acid (QA) is attenuated by acetate [2]. Ac-YVAD-CHO (i.p., 10 and 50 mg/kg for one hour). Acetate is quickly removed from the circulation and drops dramatically to between 1 and 0.2 μM at 30 and 60 minutes after injection [2].

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A potent, reversible, tetrapeptide inhibitor of interleukin-1 beta converting enzyme (ICE), L-709,049, has been shown to suppress the in vitro production of mature IL-1 beta. We now report that this inhibitor also effectively suppresses the production of mature IL-1 beta in a murine model of endotoxic shock. Intraperitoneal administration of L-709,049 reduced the elevations of IL-1 beta in the plasma and peritoneal fluid of mice treated with LPS in a dose-related manner (ED50 = 2 +/- 0.9 mg/kg). LPS-induced elevations in IL-1 alpha and IL-6 in these mice were unaffected, indicating that the inhibitor specifically affected IL-1 beta production. Immunoblot analysis of plasma and peritoneal fluid indicated that L-709,049 suppressed the formation of mature IL-1 beta production in vivo. When mouse blood was incubated in vitro with LPS, IL-1 beta was released into the plasma. This assay was used to determine ex vivo the activity of an ICE inhibitor in the blood following its administration to mice. Blood obtained 15 minutes after ip administration of 10 mg/kg of L-709,049 to mice produced 80% less IL-1 beta than control blood, and IL-1 beta production returned to control levels in blood obtained 30 minutes after injection of this inhibitor. In addition, the capacity of the blood plasma obtained from these animals to prevent the cleavage of a synthetic substrate by ICE disappeared within 1 h of ip administration of 50 mg/kg of inhibitor. [1]

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Pre-treatment with Ac-YVAD-CHO inhibited QA-induced Internucleosomal DNA fragmentation. Ac-YVAD-CHO inhibited QA-induced increases in caspase-1 activity and p53 protein levels, but had no effect on QA-induced IκB-α degradation, NF-κB or AP-1 activation. Conclusion: Caspase-1 is involved in QA-induced p53 upregulation but not IκB-α degradation. Inhibition of caspase-1 attenuates QA-induced apoptosis in rat striatum [2].

Induction of apoptosis in a cell-free reconstitution system [3]
This procedure was conducted as previously described. The reaction mixture contained 40 μl of S-100 fraction (∼5 mg/ml); 10 μl of nuclei solution (∼1 × 106 nuclei); and 400 ng of pretreated rh-caspase-1 or rh-caspase-3, 15.6 μM Ac-YVAD-CHO, or 15.6 μM Ac-DEVD-cho in the final concentration, in a final volume of 80 μl of buffer F (10 mM HEPES (pH 7.4), 40 mM b-glycerophosphate, 50 mM NaCl, 2 mM MgCl2, 4 mM EGTA, 2 mM ATP, 10 mM creatinine phosphate, 50 μg/ml creatinine kinase, and 0.2 mg/ml BSA). The mixtures were incubated at 37°C for 140 min and were occasionally mixed. The reaction solution was then mixed with 500 μl of buffer G (50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1% SDS, and 0.2 mg/ml of proteinase K) and incubated at 37°C for 1 h. The solution was extracted with phenol/chloroform. DNA isolation and electrophoresis were conducted as described previously.

Western Blot Analysis[3]
Cell Types: SNAP-treated thymocytes
Tested Concentrations: 15.6 μM
Incubation Duration: 12 h
Experimental Results: decreased PARP cleavage.

Animal/Disease Models: P. acnes-sensitized mice[1]
Doses: 50 mg/kg
Route of Administration: Ip
Experimental Results: Suppressed IL-1β levels in blood.

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Animal/Disease Models: Quinolinic acid-treated Rats[2]
Doses: 2-8 μg
Route of Administration: Intrastriatal infusion.
Experimental Results: Attenuated Quinolinic acid (QA)-induced increases in p53 and apoptosis in rat striatum. Inhibited QA-induced increases in caspase-1 activity and p53 protein levels, with no effect on QA-induced IκB-α degradation, NF -κB or AP-1 activation.

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Rats were pre-treated with intrastriatal infusion of Ac-YVAD-CHO (2-8 μg) before intrastriatal injection of QA (60 nmol). Striatal total proteins, genomic DNA, and nuclear proteins were isolated. The effects of Ac-YVAD-CHO on QA-induced caspase-1 activity, Internucleosomal DNA fragmentation, IκB-α degradation, NF-κB, and AP-1 activation, and increases in p53 protein levels were measured with enzyme assays, agarose gel electrophoresis, electrophoresis mobility shift assays, and Western blot analysis.[2]

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Stereotaxic drug administration Sprague-Dawley rats (300–350 g) were used. Rats were anesthetized with pentobarbital sodium (50 mg/kg). Stereotaxic drug administration was performed using a Kopf stereotaxic apparatus as described by Qin et al. To study the effects of a caspase-1 inhibitor on QA-induced internucleosomal DNA fragmentation, rats were either pretreated with an intrastriatal infusion of Ac-YVAD-CHO (2–8 µg) or Me2SO (2 µL) 10 min before instrastriatal injection of QA (60 nmol) and then killed 24 h after QA administration, or pre-treated with intrastriatal infusion of Ac-YVAD-CHO (4 µg) 10 min before instrastriatal injection of QA (60 nmol) and killed 12, 24, or 48 h after QA administration. Striatal genomic DNA was isolated and electrophoresed on a 2% agarose gel. To study the effect of a caspase-1 inhibitor on QA-induced increases in caspase-1 activity, rats were pre-treated with an intrastriatal infusion of Ac-YVAD-CHO (4 µg) or Me2SO (2 µL ) 10 min before intrastriatal injection of QA (60 nmol) and then killed 12 h after QA treatment. Striatal homogenates were used for assay of caspase-1 activity. To study the effect of a caspase-1 inhibitor on QA-induced increases in p53 proteins and NF-κB and AP-1 binding activities, rats were pre-treated with intrastriatal infusion of Ac-YVAD-CHO (4 µg) or Me2SO (2 µL) 10 min before intrastriatal injection of QA (60 nmol) and then killed 24 h after QA treatment. Total striatal proteins were extracted for Western blot analysis. Other animals were killed 12 h after QA treatment and nuclear proteins were isolated from the striatum for an electrophoresis mobility shift assay.

[1]. A synthetic inhibitor of interleukin-1 beta converting enzyme prevents endotoxin-induced interleukin-1 beta production in vitro and in vivo. J Interferon Cytokine Res. 1995;15(3):243-248.

[2]. Caspase-1 inhibitor Ac-YVAD-CHO attenuates quinolinic acid-induced increases in p53 and apoptosis in rat striatum. Acta Pharmacol Sin. 2005 Feb;26(2):150-4.

[3]. Nitric oxide induces thymocyte apoptosis via a caspase-1-dependent mechanism. J Immunol. 2000 Aug 1;165(3):1252-8.

In conclusion, we found that the caspase-1 inhibitor AcYVAD-CHO inhibited the QA-induced increase in p53 protein levels and internucleosomal DNA fragmentation, but had no effect on QA-induced IκB-α degradation and NF-κB activation. These results suggest that caspase-1 plays an important role in QA-induced p53 induction and apoptosis, but caspase-1 does not contribute to the QA-induced degradation of IκB-α or NF-κB activation. [2]

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Caspase-1 activity was four times higher in SNAP-treated thymocytes at 8 and 12 h compared with untreated cells. Caspase-1 activity decreased thereafter. Both ZVAD-fmk and Ac-YVAD-cho inhibited NO-induced apoptosis when added up to 12 h after SNAP treatment. The inhibitory effect was lost after 16 h. PARP cleavage takes place after the amino acid sequence Ac-DEVD-cho. Originally this activity was attributed to caspase-3, but caspases-2,-4,-6,-7,-8, and -10, when added at high concentrations, can also cleave PARP. Because the rate of spontaneous apoptosis in our thymocyte cultures was 20%, it is not surprising that some (mild) PARP cleavage was found in untreated thymocytes. However, there was greater PARP cleavage after SNAP treatment, and it was blocked by Ac-YVAD-cho, even when added 8 h after SNAP treatment. Because neither caspase-3 nor caspase-9 was significantly activated during SNAP treatment, caspase-1 (or other caspases) was likely responsible for the cleavage of PARP in our cultures. [3]

C25H36N4O10

552.57

552.24314336

Ac-YVAD-CHO;143313-51-3

168007106

Ac-Tyr-Val-Ala-Asp-al.CH3CO2H; N-acetyl-L-tyrosyl-L-valyl-L-alanyl-L-aspart-1-al acetic acid;

YVAD

Typically exists as solid at room temperature

7

10

13

39

811

4

C[C@@H](C(=O)N[C@@H](CC(=O)O)C=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)C.CC(=O)O

BWQDODQMGYONOO-HZUAXBBDSA-N

InChI=1S/C23H32N4O8.C2H4O2/c1-12(2)20(23(35)24-13(3)21(33)26-16(11-28)10-19(31)32)27-22(34)18(25-14(4)29)9-15-5-7-17(30)8-6-15;1-2(3)4/h5-8,11-13,16,18,20,30H,9-10H2,1-4H3,(H,24,35)(H,25,29)(H,26,33)(H,27,34)(H,31,32);1H3,(H,3,4)/t13-,16-,18-,20-;/m0./s1

(3S)-3-[[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-4-oxobutanoic acid;acetic acid

Ac-YVAD-CHO acetate; Ac-YVAD-CHO (acetate); HY-120019A; Ac-YVAD-CHO (L-709049) acetate

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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