Caspase 8

Inhibition of Caspases by Tetrapeptide Aldehydes [1]
The selection of tetrapeptide aldehydes for this study was based on an understanding of the known substrate specificity of these enzymes. The aldehydes Ac-WEHD-CHO and Ac-DEVD-CHO contain the optimal tetrapeptide recognition motif for Group I and II caspases, respectively. The peptide in Boc-IETD-CHO resembles the preferred sequences for Group III caspases. This is also the sequence found at the site of cleavage of the caspase-3 proenzyme, a likely endogenous substrate for these enzymes. Ac-YVAD-CHO was selected because it was formerly the most potent reversible caspase inhibitor known and, as a consequence, has been used extensively as a biological tool. Finally, Boc-AEVD-CHO was included as it was anticipated to be a broad specificity caspase inhibitor, based again on the known amino acid preferences of these enzymes.
Group III caspases are broadly inhibited by Boc-IETD-CHO, Ac-DEVD-CHO, and Boc-AEVD-CHO with K i values ranging from ∼1 to 300 nm. As anticipated from the substrate specificity studies, Ac-WEHD-CHO and Ac-YVAD-CHO are relatively poor inhibitors of this group of enzymes, consistent with the finding that hydrophobic amino acids in P4 are generally not well tolerated by members of this group and, in the case of Ac-YVAD-CHO, reflecting this group's stringent specificity for Glu in S3.

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These experiments were performed on PBLs pretreated with four different caspase inhibitors, the pan-caspase inhibitor ZVAD-fmk and the caspase 8 inhibitors Boc-AEVD-CHO, Ac-YVAD-cmk and AC-IETD-CHO that are relatively selective for caspase 8 at least at the used concentration (Garcia-Calvo et al., 1998). The results displayed in Figures 2a–d reveal that incubation of human PBLs with 1 μ M Ac-YVAD-cmk or Ac-IETD-CHO prevented CD95-mediated ASM translocation and activation (Figures 2a and b), ceramide release (Figure 2b) and CD95 clustering (Figures 2c and d). Control studies confirmed that Ac-YVAD-cmk and Ac-IETD-CHO also prevented CD95-induced apoptosis (not shown). Concurrently, human PBLs not treated with the caspase inhibitor readily responded to CD95 ligation (Figures 2a–d). Similarly, ZVAD-fmk and Boc-AEVD-CHO almost completely prevented ASM translocation, ASM activation and CD95 clustering (not shown). These studies support the notion that ASM activation and CD95 clustering occur downstream of initiator caspase activation [2].

Enzymatic Assays [1]
The activity of each enzyme was measured using continuous fluorometric assays analogous to those previously described for caspase-1 and caspase-3. In each case, a substrate with the general structure Ac-XEXD-AMC was employed, incorporating a peptide that is identical or similar to the optimal tetrapeptide recognition motif for each enzyme: caspase-1, -4, and -5 (Ac-WEHD-AMC); caspase-2, -3, -7, and -8 (Ac-DEVD-AMC); and caspase-6, -9, and -10 (Ac-VEHD-AMC). Briefly, appropriate dilutions of enzyme were added to reaction mixtures containing substrate (at a concentration ≤ K m) and various concentrations of the inhibitor of interest in a final reaction volume of 100 μl. Liberation of AMC was monitored continuously at room temperature using a Tecan Fluostar 96-well plate reader using an excitation wavelength of 380 nm and an emission wavelength of 460 nm. Unless otherwise indicated, all experiments were carried out at room temperature under standard reaction conditions defined as 0.1 m HEPES, 10% sucrose, 0.1% CHAPS, and 10 mm DTT, pH 7.5, at 25 °C.

To inhibit caspase 8, the cells were incubated for 20 min with 1 μ M of the relatively specific caspase 8 inhibitors Boc-AEVD-CHO, Ac-YVAD-cmk and Ac-IETD-CHO and the nonspecific caspase inhibitor Z-VAD-fmk [2].

[1]. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem. 1998 Dec 4;273(49):32608-13.

[2]. Ceramide-mediated clustering is required for CD95-DISC formation. Oncogene. 2003 Aug 21;22(35):5457-70.

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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Early events required for induction of apoptosis by CD95 are preassociation of CD95, the formation of the death-inducing signaling complex (DISC) and clustering of CD95 in distinct membrane domains. Here, we identify the molecular ordering of these events and show that the acid sphingomyelinase (ASM) functions upstream of the DISC to mediate CD95 clustering in ceramide-enriched membrane platforms, an event that is required for DISC formation. Experiments in ASM-deficient cells revealed that CD95 ligation, in the absence of ceramide generation, triggers <1% of full caspase 8 activation at the receptor. This event, however, is both necessary and sufficient to trigger translocation of ASM onto the outer leaflet of the plasma membrane, ASM activation and ceramide release, but insufficient for apoptosis induction. Ceramide-mediated CD95 clustering then amplifies the primary CD95 signaling and drives the second step of CD95 signaling, that is, formation of the DISC yielding 100% caspase activity and apoptosis. These studies suggest that the most parsimonious interpretation of the molecular ordering of the earliest events in CD95 signaling, at least in some cells, is: CD95 ligation-->1% of maximum caspase 8 activation-->ASM translocation-->ceramide generation-->CD95 clustering-->DISC formation-->100% of maximum caspase 8 activation-->apoptosis. [2]

C22H36N4O10

516.54

516.243

220094-15-5

6324621

Boc-Ala-Glu-Val-Asp-CHO; Boc-Ala-Glu-Val-Asp-al; N-tert-butoxycarbonyl-L-alanyl-L-alpha-glutamyl-L-valyl-L-aspart-1-al

AEVD; Boc-AEVD-CHO

Typically exists as solid at room temperature

1.258

1.112

6

10

16

36

837

4

CC(C)C(C(=O)NC(CC(=O)O)C=O)NC(=O)C(CCC(=O)O)NC(=O)C(C)NC(=O)OC(C)(C)C

MAMIJEIFDIXCPX-WSMBLCCSSA-N

InChI=1S/C22H36N4O10/c1-11(2)17(20(34)24-13(10-27)9-16(30)31)26-19(33)14(7-8-15(28)29)25-18(32)12(3)23-21(35)36-22(4,5)6/h10-14,17H,7-9H2,1-6H3,(H,23,35)(H,24,34)(H,25,32)(H,26,33)(H,28,29)(H,30,31)/t12-,13-,14-,17-/m0/s1

(4S)-5-[[(2S)-1-[[(2S)-1-carboxy-3-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-[[(2S)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoyl]amino]-5-oxopentanoic acid

Boc-aevd-cho; 220094-15-5; (4S)-5-[[(2S)-1-[[(2S)-1-carboxy-3-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-[[(2S)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoyl]amino]-5-oxopentanoic acid; Boc-AEVD-CHO trifluoroacetate salt; N-Boc-AEVD-CHO;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)