Aloxistatin (E-64d)
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MedKoo CAT#: 319511

CAS#: 88321-09-9

Description: Aloxistatin (E-64d), is a selective cysteine protease inhibitor or calpain and autophagy inhibitor. E-64d prevents in vitro cerulein- induced trypsinogen activation. E-64d can enter the intact cell and inhibit calpain. E-64d has been shown safe for the treatment of Alzheimer's disease in human. E-64d is potentially useful in the treatment of developmental seizure-induced brain damage both by regulating abnormal zinc signal transduction and through the modulation of altered lipid metabolism via ApoE/clusterin pathway in hippocampus.


Chemical Structure

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Aloxistatin (E-64d)
CAS# 88321-09-9

Theoretical Analysis

MedKoo Cat#: 319511
Name: Aloxistatin (E-64d)
CAS#: 88321-09-9
Chemical Formula: C17H30N2O5
Exact Mass: 342.21547
Molecular Weight: 342.436
Elemental Analysis: C, 59.63; H, 8.83; N, 8.18; O, 23.36

Price and Availability

Size Price Availability Quantity
10.0mg USD 150.0 Ready to ship
25.0mg USD 250.0 Ready to ship
50.0mg USD 450.0 Ready to ship
100.0mg USD 750.0 Ready to ship
200.0mg USD 1350.0 Ready to ship
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Synonym: E-64d; E 64d; E64d; E64-d; E64-d; E64 d; ethyl ester Loxistatin; NSC 694281; NSC694281; NSC-694281; EST; EP-453; EP453; EP 453; Aloxistatin

IUPAC/Chemical Name: ethyl (2S,3S)-3-(((S)-1-(isopentylamino)-4-methyl-1-oxopentan-2-yl)carbamoyl)oxirane-2-carboxylate

InChi Key: SRVFFFJZQVENJC-IHRRRGAJSA-N

InChi Code: InChI=1S/C17H30N2O5/c1-6-23-17(22)14-13(24-14)16(21)19-12(9-11(4)5)15(20)18-8-7-10(2)3/h10-14H,6-9H2,1-5H3,(H,18,20)(H,19,21)/t12-,13-,14-/m0/s1

SMILES Code: CCOC([C@@H]1[C@@H](C(N[C@H](C(NCCC(C)C)=O)CC(C)C)=O)O1)=O

Appearance: Solid powder

Purity: >98% (or refer to the Certificate of Analysis)

Shipping Condition: Shipped under ambient temperature as non-hazardous chemical. This product is stable enough for a few weeks during ordinary shipping and time spent in Customs.

Storage Condition: Dry, dark and at 0 - 4 C for short term (days to weeks) or -20 C for long term (months to years).

Solubility: Soluble in DMSO, not in water

Shelf Life: >2 years if stored properly

Drug Formulation: This drug may be formulated in DMSO

Stock Solution Storage: 0 - 4 C for short term (days to weeks), or -20 C for long term (months).

HS Tariff Code: 2934.99.9001

Biological target: Aloxistatin (E64d) is a cell-permeable and irreversible broad-spectrum cysteine protease inhibitor. Aloxistatin (E64d) exhibits entry-blocking effect for MERS-CoV.
In vitro activity: E-64d, a membrane permeant derivative of E-64c, a thiol protease inhibitor (Tamai et al. (1986) J. Pharmacobio-Dyn. 9, 672-677), was tested for ability to inhibit calpain activity in intact platelets. Calpain activity was measured by proteolysis of actin-binding protein and talin, two known substrates of calpain. Incubation of platelets with E-64c (not permeant) or E-64d before lysis prevented proteolysis after lysis. When the platelets were incubated with E-64c or E-64d and then washed to remove the drugs before lysis, only E-64d inhibited proteolysis. When platelets were incubated with E-64c or E-64d and then activated with A23187 plus calcium, a treatment that activates intraplatelet calpain, only E-64d inhibited proteolysis. These results indicate that E-64d can enter the intact cell and inhibit calpain. Reference: Biochem Biophys Res Commun. 1989 Jan 31;158(2):432-5. https://linkinghub.elsevier.com/retrieve/pii/S0006-291X(89)80065-8
In vivo activity: To evaluate that issue, a cysteine protease inhibitor, E64d, was orally administered to normal guinea pigs or transgenic mice expressing human AβPP, both of which express the human wild-type β-secretase site sequence. In guinea pigs, oral E64d administration caused a dose-dependent reduction of up to 92% in brain, CSF, and plasma of Aβ40 and Aβ42, a reduction of up to 50% in the C-terminal β-secretase fragment (CTFβ), and a 91% reduction in brain cathepsin B activity, but increased brain BACE1 activity by 20%. In transgenic AD mice, oral E64d administration improved memory deficits and reduced brain Aβ40 and Aβ42, amyloid plaque, brain CTFβ, and brain cathepsin B activity, but increased brain BACE1 activity. It’s concluded that E64d likely reduces brain Aβ by inhibiting cathepsin B and not BACE1 β-secretase activity and that E64d therefore may have potential for treating AD patients. Reference: J Alzheimers Dis. 2011;26(2):387-408. https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21613740/

Solubility Data

Solvent Max Conc. mg/mL Max Conc. mM
Solubility
DMSO 50.0 146.02
Ethanol 33.33 97.33

Preparing Stock Solutions

The following data is based on the product molecular weight 342.436 Batch specific molecular weights may vary from batch to batch due to the degree of hydration, which will affect the solvent volumes required to prepare stock solutions.

Recalculate based on batch purity %
Concentration / Solvent Volume / Mass 1 mg 5 mg 10 mg
1 mM 1.15 mL 5.76 mL 11.51 mL
5 mM 0.23 mL 1.15 mL 2.3 mL
10 mM 0.12 mL 0.58 mL 1.15 mL
50 mM 0.02 mL 0.12 mL 0.23 mL
In vitro protocol: 1. McGowan EB, Becker E, Detwiler TC. Inhibition of calpain in intact platelets by the thiol protease inhibitor E-64d. Biochem Biophys Res Commun. 1989 Jan 31;158(2):432-5. doi: 10.1016/s0006-291x(89)80065-8. PMID: 2537073.
In vivo protocol: 1. Hook G, Hook V, Kindy M. The cysteine protease inhibitor, E64d, reduces brain amyloid-β and improves memory deficits in Alzheimer's disease animal models by inhibiting cathepsin B, but not BACE1, β-secretase activity. J Alzheimers Dis. 2011;26(2):387-408. doi: 10.3233/JAD-2011-110101. PMID: 21613740; PMCID: PMC4317342. 2. Cheng XW, Murohara T, Kuzuya M, Izawa H, Sasaki T, Obata K, Nagata K, Nishizawa T, Kobayashi M, Yamada T, Kim W, Sato K, Shi GP, Okumura K, Yokota M. Superoxide-dependent cathepsin activation is associated with hypertensive myocardial remodeling and represents a target for angiotensin II type 1 receptor blocker treatment. Am J Pathol. 2008 Aug;173(2):358-69. doi: 10.2353/ajpath.2008.071126. Epub 2008 Jun 26. PMID: 18583318; PMCID: PMC2475774.

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1: Ni H, Ren SY, Zhang LL, Sun Q, Tian T, Feng X. 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. PubMed PMID: 23266720.

2: Oh-ye Y, Inoue Y, Moriyasu Y. 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. PubMed PMID: 22105025; PubMed Central PMCID: PMC3337184.

3: Chen Z, Yao K, Xu W, Wu R. [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. Russian. PubMed PMID: 18610834.

4: Otani T. Cysteine protease inhibitor, E-64d, prevents in vitro cerulein- induced trypsinogen activation. Gastroenterology. 1998 Sep;115(3):802. PubMed PMID: 9742009.

5: McGowan EB, Becker E, Detwiler TC. Inhibition of calpain in intact platelets by the thiol protease inhibitor E-64d. Biochem Biophys Res Commun. 1989 Jan 31;158(2):432-5. PubMed PMID: 2537073.