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J Am Chem Soc. 2012 Oct 24;134(42):17704-13. doi: 10.1021/ja307599z. Epub 2012 Oct 11.

Development of α-helical calpain probes by mimicking a natural protein-protein interaction.

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  • 1Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Abstract

We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs will be useful for future investigation of calpains, while the cross-linking technique will enable exploration of other protein-protein interactions.

PMID:
22998171
[PubMed - indexed for MEDLINE]
PMCID:
PMC3523126
Free PMC Article
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