Identification and optimization of a novel inhibitor of mitochondrial calpain 10

Calpain 10 has been localized to the mitochondria and is a key mediator of Ca(2+) induced mitochondrial dysfunction. A peptide screen followed by a series of modifications identified the homodisulfide form of CYGAK (CYGAK)(2) as an inhibitor of calpain 10 while showing no inhibitory activity against calpain 1. Methylation or truncation of the N-terminal cysteine significantly reduced the inhibitory activity of (CYGAK)(2) and inhibition was reversed by reducing agents, suggesting that CYGAK forms a disulfide with a cysteine near the active site. Data suggests CYGAK may be a P' calpain inhibitor and may achieve its specificity through this mechanism. CYGAK inhibited calpain activity in intact mitochondria, renal cells, and hepatocytes, prevented Ca(2+) induced cleavage of NDUFV2, and blocked Ca(2+) induced state III dysfunction. (CYGAK)(2) is the first P' specific calpain inhibitor and will be a valuable tool to prevent Ca(2+) induced mitochondrial dysfunction and explore the function of calpain 10.