title: 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.affiliation: Center for Cell Death, Injury, and Regeneration, Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, USA.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Smith
authors.forename: Campbell
authors.initials: C
authors.lastname: McInnes
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Whitaker
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Lindsey
authors.forename: Richard F
authors.initials: RF
authors.lastname: Comer
authors.forename: Craig C
authors.initials: CC
authors.lastname: Beeson
authors.forename: Rick G
authors.initials: RG
authors.lastname: Schnellmann
mesh_terms: D000818:Animals; D001671:Biochemistry / Q000379:methods; D002118:Calcium / Q000737:chemistry; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D015195:Drug Design; D006801:Humans; D020128:Inhibitory Concentration 50; D007668:Kidney / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry; D008958:Models, Molecular; D019301:Oleic Acid / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D010455:Peptides; D019301:Oleic Acid; D002154:Calpain; C415197:calpain 10; D025261:Mitochondrial Proton-Translocating ATPases; D003545:Cysteine; D002118:Calcium