Efficacy of novel calpain inhibitors in preventing renal cell death

Inhibitors of calpains, calcium-activated neutral proteases, protect against cell death produced by anoxia and a variety of toxicants both in vitro and in vivo. The problems with known calpain inhibitors are a lack of specificity, low membrane permeability, and/or low potency. The goal of this study was to determine the effects of seven novel dipeptide and tripeptide calpain inhibitors on calpain activity and antimycin A-induced cell death in rabbit renal proximal tubule (RPT) suspensions. We chose the compounds based on their inhibitory constants for μ- versus m-calpain, specificity of the inhibitors for calpain, and membrane permeability. Only three of the compounds inhibited calpain in RPT and were cytoprotective (Z-Leu-Phe-COOH, Z-Leu-Abu-CONH-CH₂-CH(OH)-Ph, and Z-Leu-Phe-CONH-Et). Interestingly, Z-Leu-Phe-COOEt, Z-Leu-Abu-CONH-CH₂-CH(OH)-C₆F₅, and Z-Leu-Abu-CONH-CH₂-2-quinolinyl were greater than 60% cytoprotective but did not inhibit calpain in RPT. Z-Leu-Abu-CONH(CH₂)₃-morpholine was neither cytoprotective nor inhibited calpain. Although these results suggest that six of the seven peptide calpain inhibitors are cell permeable, only three of them inhibited calpain activity in RPT and were cytoprotective. Their ability to inhibit calpain or produce cytoprotection did not correlate with their ability to selectively inhibit purified μ- or m-calpain. Thus it remains to be determined whether they inhibit μ-calpain, m-calpain, or both in RPT. These results also suggest that inhibition of other protease(s) in addition to calpains may be responsible for the cytoprotective actions of some compounds.