Calpains are proteases that catalyze the limited cleavage of target proteins in response to Ca 2؉ signaling. Because of their involvement in pathological conditions such as post-ischemic injury and Alzheimer and Parkinson disease, calpains form a class of pharmacologically significant targets for inhibition. We have determined the sequence preference for the hydrolysis of peptide substrates of the ubiquitous -calpain isoform by a peptide library-based approach using the proteolytic core of -calpain (I-II). The approach, first described by Turk et al. (Turk, B. E., Huang, L. L., Piro, E. T., and Cantley, L. C. (2001) Nat. Biotechnol. 19, 661-667), involved the digestion of an N-terminally acetylated degenerate peptide library in conjunction with Edman sequencing to determine the specificity for residues found at primed positions. The cleavage consensus for these positions was then used to design a second, partially degenerate library, to determine specificity at unprimed positions. We have improved upon the original methodology by using a degenerate peptide dendrimer for determination of specificity at unprimed positions. By using this modified approach, the complete cleavage specificity profile for I-II was determined for all positions flanking the cleaved peptide. A previously known preference of calpains for hydrophobic amino acids at unprimed positions was confirmed. In addition, a novel residue specificity for primed positions was revealed to highlight the importance of these sites for substrate recognition. The optimal primed site motif (MER) was shown to be capable of directing cleavage to a specific peptide bond. Accordingly, we designed a fluorescent resonance energy transferbased substrate with optimal cleavage motifs on the primed and non-primed sides (PLFAER). The -calpain core shows a far greater turnover rate for our substrate than for those based on the cleavage site of ␣-spectrin or the proteolytic sequence consensus compiled from substrate alignments.Calpains (clan CA, family C2 in the MEROPS data base (1)), a family of calcium-activated intracellular proteases, are found in animals, plants, and possibly bacteria (2). They convert intracellular calcium signals (3) into a proteolytic signal by catalyzing the limited cleavage of target proteins (4, 5). Among the known cellular substrates of calpain are numerous cytoskeletal proteins, as well as some receptors and integral membrane proteins like the Na ϩ /Ca 2ϩ exchanger, NCX-3 (6). Calpains must be strictly regulated because they catalyze irreversible processing in the cell. In one scenario, calpains localize to the plasma membrane under activating conditions (7,8). This placement may act to position calpains where they can respond to brief calcium influxes from the opening of calcium channels, resulting in very localized and transient activity. Deactivation of calpain can come about in several ways: binding to the endogenous calpain inhibitor calpastatin (9); autoproteolytic inactivation; or simply the dissipation of local high calcium levels. Unr...

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The progressive decline in kidney function and concomitant loss of renal 1alpha-hydroxylase (CYP27B1) in chronic kidney disease (CKD) are associated with a gradual loss of circulating 25-hydroxyvitamin D(3) (25(OH)D(3)) and 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)). However, only the decrease in 1alpha,25(OH)(2)D(3) can be explained by the decline of CYP27B1, suggesting that insufficiency of both metabolites may reflect their accelerated degradation by the key catabolic enzyme 24-hydroxylase (CYP24). To determine whether CYP24 is involved in causing vitamin D insufficiency and/or resistance to vitamin D therapy in CKD, we determined the regulation of CYP24 and CYP27B1 in normal rats and rats treated with adenine to induce CKD. As expected, CYP24 decreased whereas CYP27B1 increased when normal animals were rendered vitamin D deficient. Unexpectedly, renal CYP24 mRNA and protein expression were markedly elevated, irrespective of the vitamin D status of the rats. A significant decrease in serum 1alpha,25(OH)(2)D(3) levels was found in uremic rats; however, we did not find a coincident decline in CYP27B1. Analysis in human kidney biopsies confirmed the association of elevated CYP24 with kidney disease. Thus, our findings suggest that dysregulation of CYP24 may be a significant mechanism contributing to vitamin D insufficiency and resistance to vitamin D therapy in CKD.

Calpains are intracellular calcium-activated cysteine proteases whose unregulated proteolysis following the loss of calcium homeostasis can lead to acute degeneration during ischemic episodes and trauma, as well as Alzheimer's disease and cataract formation. The determination of the crystal structure of the proteolytic core of mu-calpain (muI-II) in a calcium-bound active conformation has made structure-guided design of active site inhibitors feasible. We present here high-resolution crystal structures of rat muI-II complexed with two reversible calpain-specific inhibitors employing cyclic hemiacetal (SNJ-1715) and alpha-ketoamide (SNJ-1945) chemistries that reveal new details about the interactions of inhibitors with this enzyme. The SNJ-1715 complex confirms that the free aldehyde is the reactive species of the cornea-permeable cyclic hemiacetal. The alpha-ketoamide warhead of SNJ-1945 binds with the hydroxyl group of the tetrahedral adduct pointing toward the catalytic histidine rather than the oxyanion hole. The muI-II-SNJ-1945 complex shows residue Glu261 displaced from the S1' site by the inhibitor, resulting in an extended "open" conformation of the domain II gating loop and an unobstructed S1' site. This conformation offers an additional template for structure-based drug design extending to the primed subsites. An important role for the highly conserved Glu261 is proposed.

The Eph family of receptor tyrosine kinases has drawn growing attention due to their role in regulating diverse biological phenomena. However, pharmacological interrogation of Eph kinase function has been hampered by a lack of potent and selective Eph kinase inhibitors. Here we report the discovery of compounds 6 and 9 using a rationally designed kinase-directed library which potently inhibit Eph receptor tyrosine kinases, particularly EphB2 with cellular EC 50 s of 40 nM. Crystallographic data of EphA3 and EphA7 in complex with the inhibitors show that they bind to the "DFG-out" inactive kinase conformation and provide valuable information for structure-based design of second generation inhibitors.The Eph/ephrin family is the largest among tyrosine kinases and is unique in that the ligands and receptors are both membrane bound providing the possibility for bidirectional cell-cell signaling. Genome analysis reveals that there are 14 Eph receptors and eight ephrin ligands. Eph receptor signaling is responsible for arguably the most diverse set of biological phenomena of any tyrosine kinase family including organ development, tissue remodeling, neuronal signaling, insulin secretion, and bone metabolism. 1, 2 Not surprisingly, deregulation of ephrindependent signaling has been implicated in pathological conditions related to all of these systems. 3 The involvement of Eph/ephrin-signaling in tumorigenesis has been the most extensively investigated due to frequent upregulation of Eph receptor or ligand expression in numerous tumor types. [4][5][6] The emerging picture is complicated by the diversity of biological function that is associated with individual receptors and ligands, including oncogenic or tumor suppressor functions. The possibility of targeting Eph/ephrins therapeutically may be the most straightforward in the context of inhibiting Eph/ephrin-signaling in the vasculature as a means of preventing tumor angiogenesis. 2,6 Currently only a few small molecule Eph kinase © 2009 Elsevier Ltd. All rights reserved. * Corresponding Authors: Sirano Dhe-Paganon, Tel: 416-946-3876, E-mail: sirano.dhepaganon(@)utoronto.ca Nathanael Gray, Tel: 617-582-8590, E-mail: nathanael_gray(@)dfci.harvard.edu . † These authors contributed equally to this work Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ¶ Once inside PyMol, press the play button (at the bottom right) in order to start the 3D movie NIH Public Access To date the majority of investigation into functions of Eph receptor tyrosine kinases has been accomplished using genetic and biochemical methods. Pharmacological app...

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 nonnatural 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 -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 -calpain inactivated by the epoxide compounds WR ␥-cyano-␣-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 ␥-cyano-␣-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.

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Calpains are intracellular cysteine proteases that catalyze the cleavage of target proteins in response to Ca 2+ signaling. When Ca 2+ homeostasis is disrupted, calpain over-activation causes unregulated proteolysis, which can contribute to diseases such as post-ischemic injury and cataract formation. Potent calpain inhibitors exist, but of these many cross-react with other cysteine proteases and will need modification to specifically target calpain. Here, we present crystal structures of rat calpain 1 protease core (μI-II) bound to two α-ketoamide-based calpain inhibitors containing adenyl and piperazyl primed-side extensions. An unexpected aromatic-stacking interaction is observed between the primed-side adenine moiety and the Trp298 side chain. This interaction increased the potency of the inhibitor towards μI-II and heterodimeric m-calpain. Moreover, stacking orients the adenine such that it can be used as a scaffold for designing novel primed-side address regions, which could be incorporated into future inhibitors to enhance their calpain specificity.In response to Ca 2+ signaling, the calpain family of intracellular cysteine proteases catalyzes the limited cleavage of target proteins, resulting in changes to processes such as gene expression, cytoskeleton remodeling and apoptosis. 1 Problems arise following ischemic or cerebral injury, when cells lose their ability to regulate Ca 2+ influx to the cytoplasm. The elevated Ca 2+ concentration leads to calpain hyperactivation, which causes uncontrolled proteolysis and irreversible cell damage. Since their overactivation has been linked to the development of pathological conditions such as stroke, Alzheimer disease, Duchenne muscular dystrophy and cataractogenesis, calpains represent an important class of targets for pharmacological inhibition. 2,3To date, all known calpain isoforms are multidomain enzymes, 4 with a catalytic cleft located at the interface between domains I and II. 5 These two domains, which encompass the enzyme's proteolytic core, must each bind one Ca 2+ ion to facilitate the rearrangement of the catalytic triad and substrate binding pocket into an active conformation. 6 Although the various other domains also contribute somewhat to calpain activation, the susceptibility of full-length calpain to autolysis, subunit dissociation and aggregation following Ca 2+ activation has complicated its study in the full-length form. 7 The protease core though, remains resistant to autolysis and maintains its Ca 2+ -dependent activity, albeit, at a significantly reduced level. 8 In addition, because of the relative ease with which they can be expressed in E. coli and crystallized, these *To whom correspondence should be addressed: Department of Biochemistry, Queen's University, Kingston, ON, CANADA K7L 3N6. Tel: 613-533-6821; Fax: 613-533-2497; E-mail: robert.campbell@queensu.ca. c Current address: Cytochroma Inc., 330 Cochrane Drive, Markham, ON, Canada L3R 8E4 PDB: coordinates and structure factors for the complexes of the protease core of μ-cal...