Caspase-1 (Ki = 56 pM)

The two caspase inhibitors included in the panel were the reversible aldehyde Ac-WEHD-CHO, which is known to be very selective for caspase-1 (e.g., ∼30,000-fold lower Ki than for caspase-3) and the pan-caspase irreversible inhibitor Z-VAD-fmk, which nonselectively inhibits all the caspases, including caspase-1. As shown in Figure 6A, Ac-WEHD-CHO and Z-VAD-fmk were able to efficiently suppress the bioluminescent signal, whereas none of the other inhibitors prevented the hydrolysis of CM-269.

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Inhibition of ICE by Ac-WEHD-CHO is characterized by a relatively slow association rate constant, 2.6x l@ M-Is+, and a dissociation rate constant of 1.5x 10” s-1, corresponding to an overall dissociation constant (Kj= k,r,fk,,,) of .56pM, making it among the rno~t potent reversible inhibitors described for any caspasc. This rcprcsents an improvement in binding affinity of 14.fold over Ac-YVAD-CI-IO, the compound previously thought to be the best peptidebased, reversible inhibitor of ICE. Ac-WEHD-CHO is a more potent inhibitor than AC-WAD-CHO because there is an increase in the lifcrimc of the enzyme-inhibitor complex (tlizvvnn=0.66h, Q,~~,~,,~= 12.8h) [2].

Measurement of Ki for Ac-WEHD-CHO: [2]
The ccntinucus, fluorcmetric assay employed for these studies has been previously described. All reactions were performed under standard reaction ccnditicns (standard reaction conditions) using hcmcgenecus enzyme. Reactions were monitored continuously in a Gilford Flucro-IV flucrometer using an excitaticn wavelength of 380nM and ?p emission wevelength of 480nm. To meeeure the association rate constant (kc”), enzyme was added to reaction mixtures containing, 1 ‘Km AoYVADAMC and various ccncentrations of inhibitor. The dissociation rate ccnstant (kcff) wee determined by preincubaticn of enzyme and inhibitor (25 nM ICE, t 25 nM Ac-WEHD-CHO). followed by 500.fold diluticn into a reaction mixture containing 1OO’Km levels of substrate. The cverall dissociation constant. Ki. was calculated from the observed rates cf association and dissociation according tc the equations devel. cped by Morrison for analysis of slew and tight-binding inhibitcrs [2].

Inhibition Study of CM-269 in Cell Lysates [1]
PMA-stimulated U937 and THP-1 cell lysates (75 μg of total cell protein) were incubated with 1% DMSO, 30 μM inhibitor Z-VAD-fmk, 30 μM Ac-WEHD-CHO, 30 μM E-64, 1 mM PMSF, 1 μM Pepstatin A, or 5 mM EDTA for 1 hr at 25°C. The cleavage of CM-269 was determined as described above.

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UV-Induced Apoptosis in HeLa cells [1]
HeLa cells were cultured in DMEM with 10% FBS at 37°C in a humidified incubator with a 5% CO2, 95% air atmosphere. Two days before the experiment, cells were seeded at a density of 5.0 × 105 cells/ml into 6-cm culture dishes. UV-irradiation-induced apoptosis was initiated by exposing the cells to UVC light for 45 s. After 18 hr incubation, whole-cell lysates were prepared as described above. Equal amounts of whole-cell lysates (75 μg of total cell protein) were measured for activity toward Ac-DEVD-AFC and CM-269 under the conditions described above.

[1]. Kindermann M, et al. Selective and sensitive monitoring of caspase-1 activity by a novel bioluminescent activity-based probe. Chem Biol. 2010 Sep 24;17(9):999-1007.
[2]. Rano TA, et al. A combinatorial approach for determining protease specificities: application to interleukin-1beta converting enzyme (ICE). Chem Biol. 1997 Feb;4(2):149-55.

The role of caspase-1 in inflammation has been studied intensely over recent years. However, the research of caspase-1 has remained difficult mainly due to the lack of sensitive and selective tools to monitor not only its abundance but also its activity. Here we present a bioluminescent activity-based probe (ABP) for caspase-1, developed by the Reverse Design concept, where chemically optimized protease inhibitors are turned into selective substrate ABPs. The probe exhibits excellent selectivity for caspase-1 and ∼1000-fold increase in sensitivity compared to available fluorogenic peptidic caspase-1 substrates. Moreover, we have been able to monitor and quantify specific caspase-1 activity directly in cell lysates. The activity correlated well with processing of prointerleukin-1β and prointerleukin-18 in phorbol 12-myristate 13-acetate (PMA)-stimulated cells. A detectable caspase-1 activity was present also in nonstimulated cells, consistent with processing of constitutively expressed prointerleukin-18. [1]

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Background: Interleukin-1beta converting enzyme (ICE/caspase-1) is the protease responsible for interleukin-1beta (IL-1beta) production in monocytes. It was the first member of a new cysteine protease family to be identified. Members of this family have functions in both inflammation and apoptosis.
Results: A novel method for identifying protease specificity, employing a positional-scanning substrate library, was used to determine the amino-acid preferences of ICE. Using this method, the complete specificity of a protease can be mapped in the time required to perform one assay. The results indicate that the optimal tetrapeptide recognition sequence for ICE is WEHD, not YVAD, as previously believed, and this led to the synthesis of an unusually potent aldehyde inhibitor, Ac-WEHD-CHO (Ki = 56 pM). The structural basis for this potent inhibition was determined by X-ray crystallography.
Conclusions: The results presented in this study establish a positional-scanning library as a powerful tool for rapidly and accurately assessing protease specificity. The preferred sequence for ICE (WEHD) differs significantly from that found in human pro-interleukin-1beta (YVHD), which suggests that this protease may have additional endogenous substrates, consistent with evidence linking it to apoptosis and IL-1alpha production.[2]

C28H33N7O9

611.60

611.233

189275-71-6

5311140

Ac-Trp-Glu-His-{Asp-Aldehyde}; Ac-Trp-Glu-His-Asp-al; N-acetyl-L-tryptophyl-L-alpha-glutamyl-L-histidyl-L-aspart-1-al

WEHD

Typically exists as solid at room temperature

1.4±0.1 g/cm3

1250.2±65.0 °C at 760 mmHg

709.9±34.3 °C

0.0±0.3 mmHg at 25°C

1.633

-0.5

8

10

17

44

1070

4

CC(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C=O)CC(O)=O)=O)CC1=CN=CN1)=O)CCC(O)=O)=O)CC2=CNC3=CC=CC=C23)=O

ZSZYUXBVDPGGGX-QGQQZZQASA-N

InChI=1S/C28H33N7O9/c1-15(37)32-22(8-16-11-30-20-5-3-2-4-19(16)20)28(44)34-21(6-7-24(38)39)26(42)35-23(9-17-12-29-14-31-17)27(43)33-18(13-36)10-25(40)41/h2-5,11-14,18,21-23,30H,6-10H2,1H3,(H,29,31)(H,32,37)(H,33,43)(H,34,44)(H,35,42)(H,38,39)(H,40,41)/t18-,21-,22-,23-/m0/s1

(4S)-4-[[(2S)-2-acetamido-3-(1H-indol-3-yl)propanoyl]amino]-5-[[(2S)-1-[[(2S)-1-carboxy-3-oxopropan-2-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid

Ac-WEHD-CHO; 189275-71-6; Ac-Trp-Glu-His-Asp-aldehyde (pseudo acid); Ac-Trp-Glu-His-Asp-Aldehyde; (4S)-4-[[(2S)-2-acetamido-3-(1H-indol-3-yl)propanoyl]amino]-5-[[(2S)-1-[[(2S)-1-carboxy-3-oxopropan-2-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid; MFCD01318860; Acetyl-tryptyl-glutamyl-histidyl-aspartal; SCHEMBL1548820;

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.)