calpain and Leukemia--T-Cell

Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of stroke. In this paper, we report on a series of potent dipeptide fluoromethyl ketone inhibitors of recombinant human calpain I (rh calpain I). SAR studies revealed that while calpain I tolerates a variety of hydrophobic groups at the P1 site, Leu at P2 is preferred. However, the nature of the N-terminal capping group has a significant effect on the inhibitory activity of this series of compounds. Compound 4e [(1,2,3,4-tetrahydroisoquinolin-2-yl)carbonyl-Leu-D,L-Phe-CH2F+ ++], having a tetrahydroisoquinoline containing urea as the N-terminal capping group, is the most potent dipeptide fluoromethyl ketone inhibitor of calpain I (with a second-order rate constant for inactivation of 276,000 M-1 s-1) yet reported; tripeptide 4k (Cbz-Leu-Leu-D,L-Phe-CH2F) is equipotent. A number of compounds presented in this study displayed excellent selectivity for calpain I over cathepsins B and L, two related cysteine proteases. Compounds which exhibited good inhibitory activity in the assay against isolated rh calpain I also inhibited intracellular calpain I in a human cell line. Thus, in an intact cell assay, compounds 4e and 4k inhibited calpain I with IC50 values of 0.2 and 0.1 microM, respectively. Finally, we also disclose the first example of fluorination of a dipeptide enol silyl ether to generate the corresponding dipeptide fluoromethyl ketone.

Topics: Calpain; Cathepsin B; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptides; Endopeptidases; Humans; Hydrocarbons, Fluorinated; Ketones; Leukemia, T-Cell; Recombinant Proteins; Tumor Cells, Cultured

Whether calcium is the only major intracellular activator of calpain has not yet been established. Here we demonstrate that polyphosphoinositides may play critical roles in the activation process of mu-calpain. Experiments with purified enzyme, substrate (fodrin), and phospholipids show that only polyphosphoinositides but not other lipids significantly promote calpain action in the physiological intracellular calcium range of 10(-7) to 10(-6) M. The effect of polyphosphoinositide is exerted through both a reduction in the calcium concentration required for calpain autolysis and an increase in the Vmax of the proteolytic reaction. Neomycin, a polyphosphoinositide-binding antibiotic, inhibits both polyphosphoinositide-assisted proteolysis in test tubes and calcium-induced calpain activation coupled with substrate proteolysis in intact cells. This implies that the presence of polyphosphoinositides may actually be a prerequisite for calpain activation inside cells.

Topics: Amino Acid Sequence; Antibodies; Calcium; Calpain; Carrier Proteins; Enzyme Activation; Humans; Isoenzymes; Kinetics; Leukemia, T-Cell; Macromolecular Substances; Membrane Proteins; Microfilament Proteins; Molecular Sequence Data; Neomycin; Peptides; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipids; Tumor Cells, Cultured