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.