Abstract
Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)- or (R,R)-epoxide stereochemistries, and incorporate both natural and non-natural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m- and mu-calpain rapidly (k(2)/K(i) = 131,000 and 16,500 m(-1) s(-1), respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat mu-calpain inactivated by the epoxide compounds WR gamma-cyano-alpha-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR gamma-cyano-alpha-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.
Highlights
The use of mini-calpains as surrogates for the whole enzyme circumvents many of the problems that make the full-length enzymes difficult with which to work
The development of calpain-specific inhibitors used to lessen the damage done in reperfusion injury after ischemic episodes and in longer term pathologies, such as neurodegeneration and cataract formation, has yielded limited results in the past. This task is made difficult by the ability of the active site of calpain to accommodate a wide variety of residues in all subsites except P2, where its specificity is similar to other related cysteine proteases [23, 55]
The propensity for hydrophobic amino acids, such as Ile and Val, at the P2 position typical of clan CA proteases like calpains and papain is apparent from these specificity profiles
Summary
Mini-calpains, recombinant fragments of the full-length enzyme composed exclusively of the proteolytic domains I and II, can be used as surrogates to study the proteolytic activity of calpains [21,22,23,24]. Libraries of peptidyl compounds can be made with a variety of both natural and nonnatural amino acids in the recognition region using combinatorial peptide-based solid-phase synthesis We have applied such a library of positional scanning epoxide-based compounds in conjunction with a mediumthroughput SDS-PAGE-based screening assay to profile m-calpain, the mini-calpains I–II and mI–II engineered with the stabilizing mutation G203A, and the related cysteine protease papain. These preference profiles demonstrate the differences in the specificity between the proteases, including some differences in the active-site specificity between mini and full-length calpains. The most calpain-specific compound, WRH(R,R), was demonstrated to exclusively inactivate calpain irreversibly, supporting the usefulness of positional scanning libraries in the design of inhibitors and leads for activity-based probes
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