Calpain 10 homology modeling with CYGAK and increased lipophilicity leads to greater potency and efficacy in cells.

Abstract

Calpain 10 is a ubiquitously expressed mitochondrial and cytosolic Ca(2+)-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 IC(50)'s of 70, 90, and 875 nM, respectively. Using isolated whole renal mitochondria CYGAK, CYGAK-OC, and CYGAK-ON had IC(50)'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, and concentrations up to 100 μM had no additional effect. RPTC treated with 10 μM CYGAK-OC for 24 h induced accumulation of ATP synthase β and NDUFB8, two calpain 10 substrates. In summary, we used molecular modeling to improve the potency of CYGAK, while creating CYGAK-oleic acid heterodimers to improve efficacy in cells. Since calpain 10 has been implicated in type 2 diabetes and renal aging, the use of this inhibitor may contribute to elucidating the role of calpain 10 in these and other diseases.

• Authors: Matthew A Smith, Campbell McInnes, Ryan M Whitaker, Christopher C Lindsey, Richard F Comer, Craig C Beeson, Rick G Schnellmann
• Journal: ACS chemical biology (ACS Chem Biol) Vol. 7 Issue 8 Pg. 1410-9 (Aug 17 2012) ISSN: 1554-8937 [Electronic] United States
• PMID: 22612451 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Peptides
• Oleic Acid
• Calpain
• calpain 10
• Mitochondrial Proton-Translocating ATPases
• Cysteine
• Calcium

Topics

• Animals
• Biochemistry (methods)
• Calcium (chemistry)
• Calpain (antagonists & inhibitors, chemistry)
• Cysteine (chemistry)
• Cytosol (metabolism)
• Dimerization
• Drug Design
• Humans
• Inhibitory Concentration 50
• Kidney (metabolism)
• Mitochondria (metabolism)
• Mitochondrial Proton-Translocating ATPases (chemistry)
• Models, Molecular
• Oleic Acid (chemistry)
• Peptides (chemistry)
• Protein Binding
• Protein Conformation
• Protein Structure, Tertiary
• Rats