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Biological Description

Description: Aloxistatin is an inhibitor of cysteine protease with blood platelet aggregation inhibiting activity. Aloxistatin is an irreversible, membrane-permeable inhibitor of lysosomal and cytosolic cysteine proteases with the ability to inhibit calpain activity in intact platelets.

In vitro:  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.

In vivo: Aloxistatin (100 mg/kg, p.o.) strongly inhibits the cathepsin B&L activities in the skeletal muscle, heart and liver of hamsters. [1] In spinal cord injury (SCI) rats, Aloxistatin provides neuroprotection in SCI lesion and penumbra. Aloxistatin reduces brain amyloid-β and improves memory deficits in Alzheimer's disease animal models by inhibiting cathepsin B activity.

Kinase Assay: The 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 Research: Aloxistatin (E64d) is dissolved in DMSO and stored, and then diluted with appropriate medium (final DMSO 0.1 %) before use. Cell proliferation and apoptosis are assessed by staining for a proliferation marker, Ki67, or an apoptotic marker, cleaved caspase 3, following the protocol described above for the polarity markers. MCF10 variants are grown in 3D rBM overlay cultures for 4 days and are treated with 0.1 % DMSO, 5 μM CA074Me or 5 μM Aloxistatin. The percentage of structures that are positive for Ki67 or cleaved caspase 3 is determined by counting a total of 100 structures on two separate coverslips with a Zeiss Axiophot epifluorescent microscope. Structures are considered Ki67 positive if they contained at least one cell staining for Ki67. Structures are considered to be caspase 3 positive if they contained at least one cell that is positive for cleaved caspase 3 and the positive cell(s) is not localized in the center of a developing lumen.

Chemical Properties

Molecular Weight: 342.436

Formula: C17H30N2O5

CAS No.: 88321-09-9

Catalog #: T6040

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References and Literature

1. Tamai M, et al. J Pharmacobiodyn. 1986, 9(8), 672-677.

2. McGowan EB, et al. Biochem Biophys Res Commun. 1989, 158(2), 432-435.

3. Murray EJ, et al. Metabolism. 1997 , 4. Ray SK, et al. Ann N Y Acad Sci. 2001, 939, 436-449.46(9), 1090-1094.

4. Ray SK, et al. Ann N Y Acad Sci. 2001, 939, 436-449.

5. Hook G, et al. J Alzheimers Dis. 2011, 26(2), 387-408.