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Biochim Biophys Acta. 2014 Dec;1840(12):3367-73. doi: 10.1016/j.bbagen.2014.08.014. Epub 2014 Sep 4.

Allosteric inhibitors of calpains: Reevaluating inhibition by PD150606 and LSEAL.

Author information

  • 1Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada. Electronic address: kristin.low@queensu.ca.
  • 2Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada. Electronic address: karunans@ucalgary.ca.
  • 3Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada. Electronic address: peter.davies@queensu.ca.
  • 4Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada. Electronic address: robert.campbell@queensu.ca.

Abstract

BACKGROUND:

The mercaptoacrylate calpain inhibitor, PD150606, has been shown by X-ray crystallography to bind to a hydrophobic groove in the enzyme's penta-EF-hand domains far away from the catalytic cleft and has been previously described as an uncompetitive inhibitor of calpains. The penta-peptide LSEAL has been reported to be an inhibitor of calpain and was predicted to bind in the same hydrophobic groove. The X-ray crystal structure of calpain-2 bound to its endogenous calpain inhibitor, calpastatin, shows that calpastatin also binds to the hydrophobic grooves in the two penta-EF-hand domains, but its inhibitory domain binds to the protease core domains and blocks the active site cleft directly.

METHODS:

The mechanisms of inhibition by PD150606 and LSEAL were investigated using steady-state kinetics of cleavage of a fluorogenic substrate by calpain-2 and the protease core of calpain1, as well as by examining the inhibition of casein hydrolysis by calpain and the autoproteolysis of calpain.

RESULTS:

PD150606 inhibits both full-length calpain-2 and the protease core of calpain-1 with an apparent noncompetitive kinetic model. The penta-peptide LSEAL failed to inhibit either whole calpain or its protease core in vitro.

CONCLUSIONS:

PD150606 cannot inhibit cleavage by calpain-2 of small substrates via binding to the penta-EF-hand domain.

GENERAL SIGNIFICANCE:

PD150606 is often described as a calpain-specific inhibitor due to its ability to target the penta-EF-hand domains of calpain, but we show that it must be acting at a site on the protease core domain instead.

KEYWORDS:

Allosteric inhibitor; Calcium-activated protease; Calpain; Enzyme kinetics; PD150606; Penta-EF-hand

PMID:
25196359
DOI:
10.1016/j.bbagen.2014.08.014
[PubMed]
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