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Literature summary for 3.4.22.61 extracted from

  • Wang, Z.; Watt, W.; Brooks, N.A.; Harris, M.S.; Urban, J.; Boatman, D.; McMillan, M.; Kahn, M.; Heinrikson, R.L.; Finzel, B.C.; Wittwer, A.J.; Blinn, J.; Kamtekar, S.; Tomasselli, A.G.
    Kinetic and structural characterization of caspase-3 and caspase-8 inhibition by a novel class of irreversible inhibitors (2010), Biochim. Biophys. Acta, 1804, 1817-1831.
    View publication on PubMed

Application

Application Comment Organism
drug development caspase-8 ia a target for structure-based drug design approach Homo sapiens

Crystallization (Commentary)

Crystallization (Comment) Organism
caspase-8 in complex with beta-strand urazole ring-containing irreversible peptidomimetic inhibitor compounds 4 and 9, vapour diffusion, at 4°C, mixing of 0.002 ml inhibitor solution, containing 100 mM inhibitor, with 0.1 ml protein solution containing 8.4 mg/ml caspase-8, 20 mM Tris, 100 mM DTT, pH 8.0, incubation for 60 min, mixing of 0.0025 ml of protein complex solution with 0.0025 ml of reservoir solution containing 1.0-1.1 M citrate, 0.1 M HEPES or PIPES, pH 6.5, 4°C, X-ray diffraction structure determination and analysis at 1.8 A resolution, molecular replacement Homo sapiens

Inhibitors

Inhibitors Comment Organism Structure
(3S)-3-([[2-(3-carboxypropyl)-1,3-dioxo-8-(2-phenylethyl)-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]carbonyl]amino)-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
-
Homo sapiens
(3S)-3-([[2-(3-carboxypropyl)-1,3-dioxo-8-(pyridin-3-yl)-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]carbonyl]amino)-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
-
Homo sapiens
(3S)-3-([[2-(3-carboxypropyl)-1,3-dioxo-8-[2-[(thiophen-2-ylacetyl)amino]ethyl]-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]carbonyl]amino)-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
-
Homo sapiens
(3S)-3-([[2-(3-carboxypropyl)-8-(2-[[(4-chlorophenyl)acetyl]amino]ethyl)-1,3-dioxo-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]carbonyl]amino)-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid compound-4 is binding to caspase-8 in a pocket far from the active site Homo sapiens
(3S)-3-([[2-[2-[(1H-benzimidazol-6-ylcarbonyl)amino]ethyl]-7-(cyclohexylmethyl)-1,3-dioxo-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]carbonyl]amino)-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
-
Homo sapiens
(3S)-3-([[2-[2-[(cyclohexylcarbonyl)amino]ethyl]-7-(cyclohexylmethyl)-1,3-dioxo-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]carbonyl]amino)-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
-
Homo sapiens
(3S)-3-[[(2-[4-carboxy-2-[(phenylacetyl)amino]butyl]-1,3-dioxo-2,3,5,7,8,9,10,10a-octahydro-1H-[1,2,4]triazolo[1,2-a]cinnolin-5-yl)carbonyl]amino]-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
-
Homo sapiens
(3S)-5-[(2,6-dichlorobenzoyl)oxy]-3-[([1,3-dioxo-2-[2-(1H-tetrazol-5-yl)ethyl]-2,3,5,7,8,9,10,10a-octahydro-1H-[1,2,4]triazolo[1,2-a]cinnolin-5-yl]carbonyl)amino]-4-oxopentanoic acid
-
Homo sapiens
4-[5-([(3S)-1-[(2,6-dichlorobenzoyl)oxy]-2,5-dioxohexan-3-yl]carbamoyl)-1,3-dioxo-8-(thiophen-2-yl)-5,8-dihydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-2(3H)-yl]butanoic acid
-
Homo sapiens
additional information structure-based drug design approach, synthesis of beta-strand urazole ring-containing irreversible peptidomimetic compounds. Z-VAD-fluoromethylketone and the peptidomimetic inhibitors inhibit caspase-8 via a three-step kinetic mechanism, i.e. a rapid equilibrium step, a slow-binding reversible step, and an extremely slow inactivation step, caspase-inhibitor interactions, overview. One of the caspase-8 structures also shows binding at a secondary, allosteric site, providing a possible route to the development of noncovalent small molecule modulators of caspase activity Homo sapiens
N-benzyloxycarbonyl-VAD-fluoromethylketone an irreversible caspase inhibitor Homo sapiens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information stopped-flow and steady-state kinetics Homo sapiens
0.0045
-
Ac-IETD-4-methylcoumarin 7-amide pH 7.2, 25°C Homo sapiens

Organism

Organism UniProt Comment Textmining
Homo sapiens
-
-
-

Purification (Commentary)

Purification (Comment) Organism
upon refolding and purification, mature caspase-8 yields the sequences S18PRE-VETD181 and L192SSP-FPSD286, for subunits A and B, respectively, the purified enzyme shows 85% of maximum activity Homo sapiens

Renatured (Commentary)

Renatured (Comment) Organism
upon refolding and purification, mature caspase-8 yields the sequences S18PRE-VETD181 and L192SSP-FPSD286, for subunits A and B, respectively, the purified enzyme shows 85% of maximum activity Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
Ac-IETD-4-methylcoumarin 7-amide + H2O an artificial caspase-8 substrate Homo sapiens Ac-IETD + 7-amino-4-methylcoumarin
-
?

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.2
-
assay at Homo sapiens

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
additional information
-
additional information inhibition kinetics, first-order inhibition kinetics, and two-step irreversible inhibition mechanism Homo sapiens

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.00033
-
pH 7.2, 25°C Homo sapiens 4-[5-([(3S)-1-[(2,6-dichlorobenzoyl)oxy]-2,5-dioxohexan-3-yl]carbamoyl)-1,3-dioxo-8-(thiophen-2-yl)-5,8-dihydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-2(3H)-yl]butanoic acid
0.00045
-
pH 7.2, 25°C Homo sapiens N-benzyloxycarbonyl-VAD-fluoromethylketone
0.00132
-
pH 7.2, 25°C Homo sapiens (3S)-3-([[2-(3-carboxypropyl)-8-(2-[[(4-chlorophenyl)acetyl]amino]ethyl)-1,3-dioxo-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]carbonyl]amino)-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
0.00152
-
pH 7.2, 25°C Homo sapiens (3S)-3-([[2-(3-carboxypropyl)-1,3-dioxo-8-[2-[(thiophen-2-ylacetyl)amino]ethyl]-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]carbonyl]amino)-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
0.0031
-
pH 7.2, 25°C Homo sapiens (3S)-3-([[2-(3-carboxypropyl)-1,3-dioxo-8-(pyridin-3-yl)-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]carbonyl]amino)-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
0.00354
-
pH 7.2, 25°C Homo sapiens (3S)-3-([[2-(3-carboxypropyl)-1,3-dioxo-8-(2-phenylethyl)-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]carbonyl]amino)-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
0.00357
-
pH 7.2, 25°C Homo sapiens (3S)-3-([[2-[2-[(cyclohexylcarbonyl)amino]ethyl]-7-(cyclohexylmethyl)-1,3-dioxo-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]carbonyl]amino)-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
0.00363
-
pH 7.2, 25°C Homo sapiens (3S)-5-[(2,6-dichlorobenzoyl)oxy]-3-[([1,3-dioxo-2-[2-(1H-tetrazol-5-yl)ethyl]-2,3,5,7,8,9,10,10a-octahydro-1H-[1,2,4]triazolo[1,2-a]cinnolin-5-yl]carbonyl)amino]-4-oxopentanoic acid
0.00644
-
pH 7.2, 25°C Homo sapiens (3S)-3-([[2-[2-[(1H-benzimidazol-6-ylcarbonyl)amino]ethyl]-7-(cyclohexylmethyl)-1,3-dioxo-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl]carbonyl]amino)-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
0.00927
-
pH 7.2, 25°C Homo sapiens (3S)-3-[[(2-[4-carboxy-2-[(phenylacetyl)amino]butyl]-1,3-dioxo-2,3,5,7,8,9,10,10a-octahydro-1H-[1,2,4]triazolo[1,2-a]cinnolin-5-yl)carbonyl]amino]-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid

General Information

General Information Comment Organism
evolution caspase-3 is an executioner caspase Homo sapiens