Identification and Optimization of a Novel Inhibitor of Mitochondrial Calpain 10

Abstract

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.

Calcium and Proteases

“…After being transfected into NIH-3 T3 cells, a calpain 10-green fluorescent protein (GFP) fusion protein was targeted to the mitochondria, and the N-terminal 15 amino acids of calpain 10 were found to be sufficient for mitochondrial targeting (Arrington et al, 2006).Functionally, mitochondrial calpain 10 was implicated in Ca 2+ -induced mitochondrial dysfunction as measured by mitochondrial swelling and decreased mitochondrial respiration, the latter of which was inhibited by calpeptin at submicromolar levels. Multipleproteolytic targets for mitochondrial calpain 10 were identified including NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and NADH dehydrogenase (ubiquinone) 1 beta subunit 8, ORP150 and ATP synthase subunit beta.. To validate the role of mitochondrial calpain 10, our laboratory developed a calpain 10-specific peptide inhibitor with an IC 50 of $100 nM that prevented the Ca 2+ -induced reduction in state 3 respiration and mitochondrial calpain 10 substrate cleavage (Rasbach, 2009).…”

Calpain-10 Activity Underlies Angiotensin II-Induced Aldosterone Production in an Adrenal Glomerulosa Cell Model

“…The percentage inhibition was approximately 20% of the AngII-elicited maximal response, a slightly lower degree of inhibition than was observed with the pan-calpain inhibitors, although we were somewhat limited in the dosage of CYGAK that we could test as this newly synthesized inhibitor is not commercially available (28,32). Nevertheless, this result provided evidence for a role of calpain-10 in AngII-elicited aldosterone production, although potential roles for other atypical calpains are not excluded.…”

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

“…In contrast, the equipotency of CYGAK and CYGAK-OC in mitochondrial matrix is likely the result of cleavage of the oleic acid prior to inhibition. We have previously shown that CYGAK does not inhibit calpain 1, 22 providing evidence that CYGAK is specific for calpain 10. Therefore, we determined the effects of CYGAK, CYGAK-OC, and CYGAK-ON in isolated cytosol, where calpains 1, 2, and 10 are available.…”

“…Previously we showed that CYGAK monomers quickly form homodimers in solution, and all experiments are the result of homodimer exposure. 22 Additionally, we have shown that a heterodimer ( i.e. , MeOPh-CYGAK) is a more potent calpain inhibitor than CYGAK, and thus use of a heterodimer should not greatly impact inhibition.…”

“…22 We demonstrated that CYGAK had an IC 50 of ~100 nM in mitochondrial matrix. Additionally, we showed that CYGAK had little effect on purified calpain 1 activity and was able to prevent mitochondrial calpain 10 substrate cleavage and a reduction in state 3 respiration after Ca 2+ overload.…”