Calpain 10 Homology Modeling with CYGAK and Increased Lipophilicity Leads to Greater Potency and Efficacy in Cells

Calpain 10 is a ubiquitously expressed mitochondrial and cytosolic Ca2+-regulated cysteine protease in which overexpression or knockdown leads to mitochondrial dysfunction and cell death. We previously identified a potent and specific calpain 10 peptide inhibitor (CYGAK), but it was not efficacious in cells. Therefore, we created a homology model using the calpain 10 amino acid sequence and calpain 1 3-D structure and docked CYGAK in the active site. Using this model we modified the inhibitor to improve potency 2-fold (CYGAbuK). To increase cellular efficacy, we created CYGAK-S-phenyl-oleic acid heterodimers. Using renal mitochondrial matrix CYGAK, CYGAK-OC, and CYGAK-ON had IC50's of 70, 90, and 875 nM, respectively. Using isolated whole renal mitochondria CYGAK, CYGAK-OC, and CYGAK-ON had IC50's of 95, 196, and >10,000 nM, respectively. Using renal proximal tubular cells (RPTC) in primary culture, 30 min exposures to CYGAK-OC and CYGAbuK-OC decreased cellular calpain activity approximately 20% at 1 μM, ...