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    Aloxistatin (Loxistatin; E64d, NSC-694281)
    Aloxistatin (Loxistatin; E64d, NSC-694281)

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    This product is for research use only, not for human use. We do not sell to patients.
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    InvivoChem Cat #: V0698
    CAS #: 88321-09-9Purity ≥98%

    Description: Aloxistatin (also known as E-64d, E-64c ethyl ester; EST, Loxistatin; NSC 694281) is a novel, potent, selective, irreversible, broad-spectrum and cell membrane-permeable cysteine protease inhibitor with anticoagulant activities. Aloxistatin acts by alkylating the cysteine thiol group in the catalytic site of the protease, also inhibits autophagy and lysosomal function.

    References: J Pharmacobiodyn. 1986 Aug;9(8):672-7; Biochem Biophys Res Commun. 1989 Jan 31;158(2):432-5.

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    Molecular Weight (MW)342.43
    CAS No.88321-09-9
    Storage-20℃ for 3 years in powder form
    -80℃ for 2 years in solvent
    Solubility (In vitro)DMSO: 68 mg/mL (198.6 mM)
    Water: <1 mg/mL
    Ethanol: 68 mg/mL (198.6 mM)
    Solubility (In vivo)2% DMSO+corn oil: 5 mg/mL
    SynonymsE-64c ethyl ester; E64d; E64-d; EST; EP-453; EP453; EP 453; Aloxistatin; Loxistatin; NSC694281; NSC 694281; NSC-694281; E-64d; E 64d; E64 d
    SMILES CodeCCOC([[email protected]@H]1[[email protected]@H](C(N[[email protected]](C(NCCC(C)C)=O)CC(C)C)=O)O1)=O 

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


    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

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    In Vitro

    In vitro activity: Aloxistatin (also known as E-64d, E-64c ethyl ester; Loxistatin; NSC 694281) is a potent, selective, irreversible, broad-spectrum and cell membrane-permeable cysteine protease inhibitor. Aloxistatin prevents in vitro cerulein- induced trypsinogen activation. Aloxistatin can enter the intact cell and inhibit calpain. E-64d has been shown to be safe for the treatment of Alzheimer's disease in human. Aloxistatin 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.  Aloxistatin can enter the intact platelet and inhibit proteolysis by inhibiting calpain. Aloxistatin blunts Parathyroid hormone (PTH)-induced cell proliferation and inhibits differentiation osteoblasts in vitro.

    Kinase Assay: CTLs and NK cells (0.8×106/mL) are treated with the inhibitors L1 (10-20 μM) or Aloxistatin (20-30 μM) for 24 hr at 37°C in 24-well plates. Cells are then used in 51Cr-release assays or are lysed to examine perforin in Western blots. The inhibitor is also added at the same concentration during the 4 hr reactions in some 51Cr-release assays, as indicated. Cell lysates are prepared using NP-40 lysis buffer (25 mM HEPES, 250 mM NaCl, 2.5 mM ethylenediaminetetraacetic acid, 0.1% volume/volume Nonidet P-40) and total protein concentration is determined using the Bradford assay. Equal amounts of protein are loaded and resolved on 8% SDS-PAGE gels. Human or mouse perforin is detected using the appropriate antibodies as indicated. Anti-actin antibody is used as a loading control.

    Cell Assay: 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.

    In VivoAfter 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.
    Animal modelMale Sprague–Dawley rats
    Formulation & Dosage4 μg; i.p. injection
    ReferencesJ Pharmacobiodyn. 1986 Aug;9(8):672-7; Biochem Biophys Res Commun. 1989 Jan 31;158(2):432-5; Toxicol Lett. 2013 Feb 27;217(2):162-9.

    These protocols are for reference only. InvivoChem does not independently validate these methods.






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