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E 64d

Cell-permeable, irreversible cysteine protease inhibitor

E 64d

Catalog No. A1903
Size Price Stock Qty
Evaluation Sample $28.00  All Inclusive In stock
1mg $50.00 In stock
5mg $150.00 In stock
25mg $500.00 In stock
100mg $800.00 In stock

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Sample solution is provided at 25 µL, 10mM.

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Chemical structure

E 64d

Related Biological Data

E 64d

Related Biological Data

E 64d

Biological Activity

E-64d, a synthetic analog of E-64 and ethyl ester of E-64c, is an irreversible, membrane-permeable inhibitor of lysosomal and cytosolic cysteine proteases. E-64d inhibits calpain and the cysteine proteases cathepsins F, K, B, H, and L.
Targets cathepsins F cathepsins K cathepsins B cathepsins H cathepsins L  
IC50            

Protocol

Cell experiment: [1]

Cell lines

Platelets

Preparation method

This product is soluble in ethanol (>10 mg/ml), DMSO (20 mg/ml), DMF (30 mg/ml), and 1:1 DMSO:PBS (pH 7.2) (~0.5 mg/ml). It is insoluble in water. Stock solution can be stored at -80 °C less than 6 months.

Reacting condition

50 μg/ml, 10 min

Applications

Platelets were activated with A23187 plus calcium, a condition known to lead to calpain-catalyzed proteolysis of ABP and talin. In the absence of inhibitor, A23187 led to complete degradation of ABP and talin. E 64d, the permeant inhibitor, did inhibit intracellular proteolysis. Some inhibition was observed at the lowest concentration tested, 20 μg/ml, and essentially complete inhibition was obtained with 50 μg/ml.

Animal experiment: [2]

Animal models

Male Sprague–Dawley rats

Dosage form

Intraperitoneal injection, 4 μg

Application

After E-64d treatment, mice were treated with penicillin to induce recurrent seizures. The result showed that E-64d remarkably reduced the aberrant mossy fiber sprouting in the supragranular region of dentate gyrus and CA3 subfield of hippocampus. In rats without E-64d treatment, there was prominent aggregation of mossy fiber terminals in the dentate gyrus and in the stratum pyramidale of CA3 subfield. In rats treated with E-64d, the aggregation of mossy fiber terminals was remarkably decreased in both the supragranular region of dentate gyrus and CA3 subfield.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1] McGowan E B, Becker E, Detwiler T C. Inhibition of calpain in intact platelets by the thiol protease inhibitor E-64d. Biochemical and biophysical research communications, 1989, 158(2): 432-435.

[2] Ni H, Ren S, Zhang L, et al. Expression profiles of hippocampal regenerative sprouting-related genes and their regulation by E-64d in a developmental rat model of penicillin-induced recurrent epilepticus. Toxicology letters, 2013, 217(2): 162-169.

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Chemical Properties

Cas No. 88321-09-9 SDF Download SDF
Chemical Name ethyl (2S,3S)-3-[[(2S)-4-methyl-1-(3-methylbutylamino)-1-oxopentan-2-yl]carbamoyl]oxirane-2-carboxylate
Canonical SMILES CCOC(=O)C1C(O1)C(=O)NC(CC(C)C)C(=O)NCCC(C)C
Formula C17H30N2O5 M.Wt 342.43
Solubility Soluble in DMSO Storage Store at -20°C
Shipping Condition: Evaluation sample solution : ship with blue ice
All other available size: ship with RT , or blue ice upon request

View Related Products By Research Topics

Research Update

1. Expression profiles of hippocampal regenerative sprouting-related genes and their regulation by E-64d in a developmental rat model of penicillin-induced recurrent epilepticus. Toxicol Lett. 2013 Feb 27;217(2):162-9. doi: 10.1016/j.toxlet.2012.12.010. Epub 2012 Dec 21.
Abstract
E-64d inhibited hippocampal aberrant mossy fiber sprouting and seizure-induced up-regulation of ApoE and Clusterin in rats. A significant down-regulation of PRG-1, PRG-3, prg-5, cathepsin B amd ApoE and a up-regulation of nSMase and ANX7 were observed in hippocampus of E-64d-pretreated seizure rats.
2. Detecting autophagy in Arabidopsis roots by membrane-permeable cysteine protease inhibitor E-64d and endocytosis tracer FM4-64. Plant Signal Behav. 2011 Dec;6(12):1946-9.
Abstract
In order to detect autophage, E-64d, a membrane-permeable cysteine protease inhibitor, was applied in culture medium where root tips from Arabidopsis seedlings were incubated.
3. [Inhibition of calpain expression by E-64d in the rat retina subjected to ischemia/reperfusion injury]. Mol Biol (Mosk). 2008 Mar-Apr;42(2):258-64.
Abstract
E-64d exhibits protective effects against IRI-induced retinal apoptosis, since it inhibited IRI-induced up-regulation of m-calpain expression, the crease of m-calpain/calpastatin ratio and IRI-induced retinal damage in rats.
4. Inhibition of calpain in intact platelets by the thiol protease inhibitor E-64d. Biochem Biophys Res Commun. 1989 Jan 31;158(2):432-5.
Abstract
E-64d is a membrane permeant inhibitor of calpain that enters intact cells and inhibits proteolysis once it’s incubated with platelets before lysis.
5. CB-64D and CB-184: ligands with high sigma 2 receptor affinity and subtype selectivity. Eur J Pharmacol. 1995 May 24;278(3):257-60.
Abstract
CD-64L, CB-64D, CB-182 and CB-184 exhibited sigma 1 and sigma 2 Ki of 10.5 and 154 nM, 3063 and 16.5 nM, 27.3 and35.5 nM and 7436 and 13.4 nM respectively, where CB-64D and CB-184 displayed high sigma 2 receptor affinity and subtype selectivity.

Background

E-64d, a membrane permeant derivative of E-64c, a thiol protease inhibitor1, was tested for ability to inhibit calpain activity in intact platelets.

E-64c or E-64d also inhibited (lanes 3-8), demonstrating their effect on calpain. When the platelets were incubated with these inhibitors for I0 min and were then washed to remove extracellular inhibitor before lysis, neither E-64c nor leupeptin inhibited proteolysis, but E-64d did inhibit. E-64d was able to penetrate the platelet and was thus not removed by washing.E-64c failed to inhibit proteolysis in intact platelets, but E-64d, the permeant inhibitor, did  inhibit intracellular proteolysis.E-64c and E-64d were each able to inhibit the protease activity in lysed platelets. This protease activity has been attributed to calpain by its absolute dependence on Ca 2+and by inhibition by known inhibitors of calpain. E-64d is able to enter the  intact platelet: i) after washing to remove extracellular inhibitor, there was  no protease activity detected after platelet lysis, and ii) activation of  platelets preincubated with E-64d, but not E-64c, resulted in inhibition of  proteolysis by calpain activated in intact platelets by A23187 plus calcium.

Reference:
1. M. Tamai, K. Matsumoto, S. Omura, I. Koyama, Y. Ozawa, K. Hanada J. Pharmacobio-Dyn., 9 (1986), pp. 672–677
2. E. B. McGowan, E. Becker, and T. C. Detwiler, INHIBITION OF CALPAIN IN INTACT PLATELETS BY THE THIOL PROTEASE INHIBITOR E-64d. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS , Vol. 158, No. 2, 1989