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 Ṕ 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 Ṕ specific calpain inhibitor and will be a valuable tool to prevent Ca ²⁺ induced mitochondrial dysfunction and explore the function of calpain 10.