title: Development of α-helical calpain probes by mimicking a natural protein-protein interaction.
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.
authors.affiliation: Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Hyunil
authors.initials: H
authors.lastname: Jo
authors.forename: Nataline
authors.initials: N
authors.lastname: Meinhardt
authors.forename: Yibing
authors.initials: Y
authors.lastname: Wu
authors.forename: Swapnil
authors.initials: S
authors.lastname: Kulkarni
authors.forename: Xiaozhen
authors.initials: X
authors.lastname: Hu
authors.forename: Kristin E
authors.initials: KE
authors.lastname: Low
authors.forename: Peter L
authors.initials: PL
authors.lastname: Davies
authors.forename: William F
authors.initials: WF
authors.lastname: DeGrado
authors.forename: Doron C
authors.initials: DC
authors.lastname: Greenbaum
mesh_terms: D002135:Calcium-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; C033668:calpastatin; D002154:Calpain