Allosteric inhibitors of calpains: Reevaluating inhibition by PD150606 and LSEAL

作者：Low Kristin E; Partha Sarathy Karunan; Davies Peter L; Campbell Robert L^*

来源：Biochimica et Biophysica Acta-General Subjects, 2014, 1840(12): 3367-3373.

DOI：10.1016/j.bbagen.2014.08.014

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

Background: The mercaptoacrylate calpain inhibitor, PD150606, has been shown by X-ray crystallography to bind to a hydrophobic groove in the enzyme%26apos;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. %26lt;br%26gt;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. %26lt;br%26gt;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. %26lt;br%26gt;Conclusions: PD150606 cannot inhibit cleavage by calpain-2 of small substrates via binding to the penta-EF-hand domain. %26lt;br%26gt;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.

• 出版日期2014-12