A1903-5mg | E 64d [88321-09-9] Clinisciences

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
3. Carmen JC, Sinai AP. The Differential Effect of Toxoplasma Gondii Infection on the Stability of BCL2-Family Members Involves Multiple Activities. Front Microbiol. 2011 Jan 24;2:1.

1. Cliff J. Luke, Stephanie Markovina, et al. "Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins." Commun Biol. 2022 Jan 12;5(1):47. PMID:35022507
2. Jianhui Nie, Qianqian Li, et al. "Functional comparison of SARS-CoV-2 with closely related pangolin and bat coronaviruses." Cell Discov. 2021 Apr 6;7(1):21. PMID:33824288
3. Wang LJ, Chiou JT, et al. "SIRT3, PP2A and TTP protein stability in the presence of TNF-α on vincristine-induced apoptosis of leukaemia cells." J Cell Mol Med. 2020;24(4):2552–2565. PMID:31930676
4. Dziekan, Jerzy Michal. "Molecular analysis of antimalarial therapies : from drug development to mode of action." Nanyang Technological University. 2019.
5. Maxine Bauzon, Penelope M. Drake, et al. "Maytansine-bearing antibody-drug conjugates induce in vitro hallmarks of immunogenic cell death selectively in antigen-positive target cells." OncoImmunology.Received 09 Aug 2018, Accepted 12 Dec 2018, Published online: 22 Jan 2019.
6. An H, Harper JW. "Systematic analysis of ribophagy in human cells reveals bystander flux during selective autophagy." Nat Cell Biol. 2017 Dec 11. PMID:29230017
7. Tsai YC, Kotiya A, et al. "Deubiquitinating enzyme VCIP135 dictates the duration of botulinum neurotoxin type A intoxication." Proc Natl Acad Sci U S A. 2017 Jun 27;114(26):E5158-E5166. PMID:28584101

Hide ↑↑

Physical Appearance	A solid
Storage	Store at -20°C
M.Wt	342.43
Cas No.	88321-09-9
Formula	C₁₇H₃₀N₂O₅
Solubility	insoluble in H2O; ≥17.12 mg/mL in DMSO; ≥18.5 mg/mL in EtOH
Chemical Name	ethyl (2S,3S)-3-[[(2S)-4-methyl-1-(3-methylbutylamino)-1-oxopentan-2-yl]carbamoyl]oxirane-2-carboxylate
SDF	Download SDF
Canonical SMILES	CCOC(=O)C1C(O1)C(=O)NC(CC(C)C)C(=O)NCCC(C)C
Shipping Condition	Small Molecules with Blue Ice, Modified Nucleotides with Dry Ice.
General tips	We do not recommend long-term storage for the solution, please use it up soon.

Cell experiment: [1]
Cell lines	Platelets
Preparation method	The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 °C for 10 minutes and/or shake it in the ultrasonic bath for a while.Stock solution can be stored below -20°C for several months.
Reaction Conditions	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
Applications	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.

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