A novel series of urea-based peptidomimetic calpain inhibitors

A series of peptide aldehyde derivatives in which the P(2) chiral carbon has been replaced with nitrogen were synthesized as urea-based peptidomimetic inhibitors of mu-calpain. The compounds mirrored the general SAR of peptidyl aldehyde calpain inhibitors but displayed greater selectivity for mu-calpain over cathepsin B.     

N terminus of calpain 1 is a mitochondrial targeting sequence

The ubiquitous m- and mu-calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, mu-calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of mu-calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1, which together form mu-calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic alpha-helical domain, and is distinct from the N terminus of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N terminus of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The presence of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of mu-calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.     

Cystatins as calpain inhibitors: engineered chicken cystatin- and stefin B-kininogen domain 2 hybrids support a cystatin-like mode of interaction with the catalytic subunit of mu-calpain

Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit mu- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and deltaL110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefin B by the corresponding KD2 sequence resulted in a calpain inhibitor of Ki = 188 nM. Deletion of L110, which forms a beta-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of mu-calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substrate-like cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatin-type inhibitors do not bind to the calmodulin-like domain of the small subunit of calpain, and their interaction with the mu-calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold.     

Synthesis and biological evaluation of novel piperidine carboxamide derived calpain inhibitors

Calpain inhibitors which are derived from piperidine carboxamides in the P2 region were prepared and evaluated for mu-calpain inhibition. In particular, the keto amides 11f and 11j have Ki of 30 and 9 nM and display a more than 100-fold selectivity over the closely related cysteine protease cathepsin B. Furthermore, these compounds inhibit NMDA induced convulsions in mice indicating that calpain inhibition in brain results in some anticonvulsive properties.     

Calpain is involved in the HIV replication from the latently infected OM10.1 cells

Treatment of OM10.1 cells latently infected with human immunodeficiency virus type 1 (HIV-1) with phorbol ester and calcium ionophore (A23187) induced virus replication which was blocked by N-Ac-Leu-Leu-norleucinal (ALLnL), a calpain inhibitor I, and not by lactacystin, a specific proteasome inhibitor. When the purified NF-kappa B/I kappa B complex was treated with mu-calpain, the specific DNA-binding activity was demonstrated by using electrophoretic mobility shift assay in vitro. This effect of mu-calpain was inhibited by ALLnL and calpastatin and not by lactacystin. In fact, we found that mu-calpain efficiently degraded I kappa B alpha. Furthermore, our Western blotting analysis has revealed that mu-calpain cleaves I kappa B alpha at its N-terminal and C-terminal regions that were previously reported to be involved in the interaction with NF-kappa B p65. These observations indicate that in monocyte/macrophage cells calcium signaling is involved in NF-kappa B activation through activation of calpain and thus calpain inhibitors may be effective in inhibiting the activation of latently infected HIV.     

mu-Calpain regulates receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis via NF-kappaB activation in RAW 264.7 cells

To clarify the role of calpain in the receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis, RANKL-induced calpain activation was examined by using murine RAW 264.7 cells and bone marrow-derived monocyte/macrophage progenitors. We found that calpain activity increased in response to RANKL in both cell types based on alpha-spectrinolysis and that mu-calpain, rather than m-calpain, was activated during RANKL-supported osteoclastogenesis in RAW 264.7 cells. Overexpression of mu-calpain clearly augmented RANKL-supported osteoclastogenesis in RAW 264.7 cells, thereby implicating its pivotal role in this process. Cell-permeable calpain inhibitors, including calpastatin and calpeptin, were sufficient to suppress RANKL-supported osteoclastogenesis based on decreased expression of the osteoclastogenic marker, matrix metalloproteinase 9, and the generation of tartrate-resistant acid phosphatase-positive multinucleated cells in both cell types. Calpain inhibitors suppressed NF-kappaB activation via inhibition of the cleavage of inhibitor of NF-kappaB(IkappaBalpha)in RAW 264.7 cells. Taken together, our findings suggest that mu-calpain is essential to the regulation of RANKL-supported osteoclastogenesis via NF-kappaB activation.     

Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UVr-1 cells derived from ultraviolet-sensitive human RSa cells

Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14 - 18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CTheta, Src, Bax and mu-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated mu-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, mu-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells.     

Calpain is required for normal osteoclast function and is down-regulated by calcitonin

Osteoclast motility is thought to depend on rapid podosome assembly and disassembly. Both mu-calpain and m-calpain, which promote the formation and disassembly of focal adhesions, were observed in the podosome belt of osteoclasts. Calpain inhibitors disrupted the podosome belt, blocked the constitutive cleavage of the calpain substrates filamin A, talin, and Pyk2, which are enriched in the podosome belt, induced osteoclast retraction, and reduced osteoclast motility and bone resorption. The motility and resorbing activity of mu-calpain(-/-) osteoclast-like cells were also reduced, indicating that mu-calpain is required for normal osteoclast activity. Histomorphometric analysis of tibias from mu-calpain(-/-) mice revealed increased osteoclast numbers and decreased trabecular bone volume that was apparent at 10 weeks but not at 5 weeks of age. In vitro studies suggested that the increased osteoclast number in the mu-calpain(-/-) bones resulted from increased osteoclast survival, not increased osteoclast formation. Calcitonin disrupted the podosome ring, induced osteoclast retraction, and reduced osteoclast motility and bone resorption in a manner similar to the effects of calpain inhibitors and had no further effect on these parameters when added to osteoclasts pretreated with calpain inhibitors. Calcitonin inhibited the constitutive cleavage of a fluorogenic calpain substrate and transiently blocked the constitutive cleavage of filamin A, talin, and Pyk2 by a protein kinase C-dependent mechanism, demonstrating that calcitonin induces the inhibition of calpain in osteoclasts. These results indicate that calpain activity is required for normal osteoclast activity and suggest that calcitonin inhibits osteoclast bone resorbing activity in part by down-regulating calpain activity.     

Molecular mode of action of a covalently inhibiting peptidomimetic on the human calpain protease core

Calpain is a nearly ubiquitous Ca2+-activated proteolytic enzyme whose precise physiological function is unknown. However, aberrant Ca2+ homeostasis in the course of cellular injuries and certain diseases of the CNS appears to activate calpain, in turn promoting the degradation of key cytoskeletal and membrane proteins. Hyperactive calpain has also been implicated in various aging phenomena and diseases of late life. Therefore, calpain is considered a potential therapeutic target in preventing degenerations of many kinds. Despite its potential medicinal importance, known structural information about mu-calpain is limited to that from the rat enzyme. We have determined the crystal structure of the human mu-calpain protease core (hmuI-II) containing a Gly213Ala mutation and covalently inactivated by a peptidomimetic (ZLLYCH2F) at 2.0 A resolution. The methylene carbon of the inhibitor is bound to Cys115. Additional hydrogen bonding and hydrophobic interactions between active site residues and the inhibitor, including an intermolecular antiparallel beta-sheet arrangement characteristically observed with members of the papain family of cysteine proteinases, help to stabilize the complex and orient the inhibitor. The terminal ZL portion of the inhibitor is solvent-exposed and highly flexible, and is thus not involved in specific binding interactions with the enzyme. Two large enzyme regions flanking the active site are highly flexible; they may be important in recognition of calpain's natural protein substrates and in positioning them for catalysis. The implications for drug design are discussed.     

Ritonavir does not inhibit calpain in vitro

Ritonavir, an inhibitor of HIV-1 protease, has been reported to also inhibit the Ca2+-dependent cysteine protease, calpain. We have investigated these claims with an in vitro study of the effect of ritonavir on the m-calpain and mu-calpain isoforms. Ritonavir failed to block either autolytic or hydrolytic calpain activity, but remained fully capable of inhibiting the HIV-1 protease. Any calpain-related effects of ritonavir in cells must, therefore, arise by a mechanism other than direct inhibition of calpains.     

Identification of calpain cleavage sites in the G1 cyclin-dependent kinase inhibitor p19(INK4d)

Calpains are a large family of Ca2+-dependent cysteine proteases that are ubiquitously distributed across most cell types and vertebrate species. Calpains play a role in cell differentiation, apoptosis, cytoskeletal remodeling, signal transduction and the cell cycle. The cell cycle proteins cyclin D1 and p21(KIP1), for example, have been shown to be affected by calpains. However, the rules that govern calpain cleavage specificity are poorly understood. We report here studies on the pattern of mu-calpain proteolysis of the p19(INK4d) protein, a cyclin-dependent kinase 4/6 inhibitor that negatively regulates the mammalian cell cycle. Our data show new characteristics of calpain action: mu-calpain cleaves p19(INK4d) immediately after the first and second ankyrin repeats that are structurally less stable compared to the other repeats. This is in contrast to features observed so far in the specificity of calpains for their substrates. These results imply that calpain may be involved in the cell cycle by regulating the cell cycle regulatory protein turnover through CDK inhibitors and cyclins.     

Crystal structures of calpain-E64 and -leupeptin inhibitor complexes reveal mobile loops gating the active site

The endogenous calpain inhibitor, calpastatin, modulates some patho-physiological aspects of calpain signaling. Excess calpain can escape this inhibition and as well, many calpain isoforms and autolytically generated protease core fragments are not inhibited by calpastatin. There is a need, therefore, to develop specific, cell-permeable calpain inhibitors to block uncontrolled proteolysis and prevent tissue damage during brain and heart ischemia, spinal-cord injury and Alzheimer's diseases. Here, we report the first high-resolution crystal structures of rat mu-calpain protease core complexed with two traditional, low molecular mass inhibitors, leupeptin and E64. These structures show that access to a slightly deeper, but otherwise papain-like active site is gated by two flexible loops. These loops are divergent among the calpain isoforms giving a potential structural basis for substrate/inhibitor selectivity over other papain-like cysteine proteases and between members of the calpain family.     

Mu-calpain is functionally required for alpha-processing of Alzheimer's beta-amyloid precursor protein

Alzheimer's beta-amyloid precursor protein (APP) is normally processed by an unidentified alpha-secretase. A unique feature of this protease is its high sensitivity to phorbol esters, yet the mechanism involved is unclear. We have previously reported that phorbol 12,13-dibutyrate (PDBu) activates calpain, a Ca2+-dependent protease, and PDBu-induced release of APPs (secreted APP) is sensitive to calpain inhibitors, suggesting that calpain is involved in APP alpha-processing. In the present study, we found that PDBu markedly promoted the expression of both mu- and m-calpains in cultured fibroblasts. Dose-response and time course studies revealed that mu-calpain was more sensitive to PDBu than m-calpain and the temporal course of the mu-calpain change coincides better with that of APPs release. Moreover, the stimulatory effect of PDBu on mu-calpain was selectively blocked by mu-calpain-specific siRNA (small interference RNA) and the blockage was accompanied by a concomitant decrease in APPs release. In contrast, m-calpain siRNA did not affect APPs release significantly. Measurement of amyloid beta protein (Abeta) release in the mu-calpain siRNA-treated cells indicated that Abeta40 and Abeta42 levels inversely changed in relation to APPs, and the changes in Abeta42 were more prominent than in Abeta40. Together, these data suggest that calpain, particularly mu-calpain, is a potential candidate for alpha-secretase in the regulated APP alpha-processing, and that changes in this protease can affect the outcome of the overall APP processing.     

Inhibitory effect of di- and tripeptidyl aldehydes on calpains and cathepsins

Eight different di- and tripeptidyl aldehyde derivatives, each having at its C-terminus an aldehyde analog of L-norleucine, L-methionine, or L-phenylalanine with a preceding L-leucine residue, were synthesized and tested for their inhibitory effects on several serine and cysteine endopeptidases. These compounds showed almost no inhibition of trypsin, and only weak inhibition of alpha-chymotrypsin and cathepsin H, while they exhibited marked inhibition of cathepsin B less than calpain II congruent to calpain I less than cathepsin L, being stronger in this order. The mode of inhibition of these cysteine proteinases was competitive for the peptide substrate used and inhibitor constants (Ki) were calculated from the Dixon plot. The best inhibitors found were: 4-phenyl-butyryl-Leu-Met-H for calpain I (Ki, 36 nM) and calpain II (Ki, 50 nM); acetyl-Leu-Leu-nLeu-H for cathepsin L (Ki, 0.5 nM); acetyl-Leu-Leu-Met-H for cathepsin B (Ki, 100 nM).     

Mu-calpain is involved in the regulation of TNF-alpha-induced matrix metalloproteinase-3 release in a rheumatoid synovial cell line

Calpain is secreted by intra-articular synovial cells and degrades the main components of cartilage matrix proteins, proteoglycan, and collagen, causing cartilage destruction. Matrix metalloproteinase-3 (MMP-3) has also been detected in synovial fluid and serum, and is involved in the development and progression of rheumatoid arthritis by degradation of the extracellular matrix and cartilage destruction. To investigate the relationship between calpain and MMP-3 in rheumatic inflammation, we utilized the rheumatic synovial cell line, MH7A. Tumor necrosis factor (TNF-alpha) stimulation-induced increased expression of mu-calpain, m-calpain, and MMP-3 in these cells, as well as the release of calpain and MMP-3 into the culture medium. The calpain inhibitors, ALLN (calpain inhibitor I) and calpeptin, did not affect the intracellular expression of MMP-3, but reduced the secretion of MMP-3 in a concentration-dependent manner. Down-regulation of mu- but not m-calpain by small interfering RNAs abolished TNF-alpha-induced MMP-3 release from the synovial cells. These findings suggest that calpain, particularly mu-calpain, regulates MMP-3 release by rheumatic synovial cells, in addition to exerting its own degradative action on cartilage.     

Effect of Ca2+ on binding of the calpains to calpastatin

Autolyzed mu-calpain, unautolyzed mu-calpain, autolyzed m-calpain, and unautolyzed m-calpain (mu-calpain is the micromolar Ca2+-requiring proteinase, m-calpain is the millimolar Ca2+-requiring proteinase) were passed through a calpastatin-affinity column at different free Ca2+ concentrations, and binding of the calpains to calpastatin was compared with proteolytic activity of that calpain at each Ca2+ concentration. Unautolyzed m-calpain, autolyzed m-calpain, and autolyzed mu-calpain required less Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Unautolyzed mu-calpain, however, required slightly more Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Half-maximal binding of oxidatively inactivated mu- or m-calpain to calpastatin required approximately the same Ca2+ concentrations as half-maximal binding of unautolyzed mu- or m-calpain, respectively, to calpastatin. Binding of unautolyzed m-calpain and autolyzed mu-calpain to calpastatin occurred over a wide range of Ca2+ concentrations, and it seems likely that two or more Ca2+-binding sites with different Ca2+-binding constants are involved in binding of the calpains to calpastatin. Proteolytic activity occurs at different Ca2+ concentrations than calpastatin binding, suggesting a second set of Ca2+-binding sites associated with proteolytic activity. Third and fourth sets of Ca2+-binding sites may be involved in autolysis and in binding to phosphatidylinositol or cell membranes; these four Ca2+-dependent properties of the calpains may require the eight potential Ca2+-binding sites that amino acid sequences predict are present in the calpain molecules.     

Degradation of p21cip1 in cells productively infected with human cytomegalovirus

Human cytomegalovirus (HCMV) stimulates arrested cells to enter the cell cycle by activating cyclin-dependent kinases (Cdks), notably Cdk2. Several mechanisms are involved in the activation of Cdk2. HCMV causes a substantial increase in the abundance of cyclin E and stimulates translocation of Cdk2 from the cytoplasm to the nucleus. Further, the abundance of the Cdk inhibitors (CKIs) p21cip1/waf1 (p21cip1) and p27kip1 is substantially reduced. The activity of cyclin E/Cdk2 increases as levels of CKIs, particularly p21cip1, fall. We have previously shown that these phenomena contribute to priming the cell for efficient replication of HCMV. In this study, the mechanisms responsible for the decrease in p21cip1 levels after HCMV infection were investigated by measuring p21cip1 RNA and protein levels in permissive human lung (LU) fibroblasts after HCMV infection. Northern blot analysis revealed that p21cip1 RNA levels increased briefly at 3 h after HCMV infection and then decreased to their nadir at 24 h; thereafter, RNA levels increased to about 60% of the preinfection level. Western blot analysis demonstrated that the relative abundance of p21cip1 protein roughly paralleled the observed changes in initial RNA levels; however, the final levels of protein were much lower than preinfection levels. After a transient increase at 3 h postinfection, p21cip1 abundance declined sharply over the next 24 h and remained at a very low level through 96 h postinfection. The disparity between p21cip1 RNA and protein levels suggested that the degradation of p21cip1 might be affected in HCMV-infected cells. Treatment of HCMV-infected cells with MG132, an inhibitor of proteasome-mediated proteolysis, provided substantial protection of p21cip1 in mock-infected cells, but MG132 was much less effective in protecting p21cip1 in HCMV-infected cells. The addition of E64d or Z-Leu-Leu-H, each an inhibitor of calpain activity, to HCMV-infected cells substantially increased the abundance of p21cip1 in a concentration-dependent manner. To verify that p21cip1 was a substrate for calpain, purified recombinant p21cip1 was incubated with either m-calpain or mu-calpain, which resulted in rapid proteolysis of p21cip1. E64d inhibited the proteolysis of p21cip1 catalyzed by either m-calpain or mu-calpain. Direct measurement of calpain activity in HCMV-infected LU cells indicated that HCMV infection induced a substantial and sustained increase in calpain activity, although there was no change in the abundance of either m- or mu-calpain or the endogenous calpain inhibitor calpastatin. The observed increase of calpain activity was consistent with the increases in intracellular free Ca2+ and phospholipid degradation in HCMV-infected LU cells reported previously from our laboratory. Considered together, these results suggest that the increase in calpain activity observed following HCMV infection contributes significantly to the reduction of p21cip1 levels and the resultant cell cycle progression.     

Mitochondrial localization of mu-calpain

Calcium-dependent cysteine proteases, calpains, have physiological roles in cell motility and differentiation but also play a pathological role following insult or disease. The ubiquitous calpains are widely considered to be cytosolic enzymes, although there has been speculation of a mitochondrial calpain. Within a highly enriched fraction of mitochondria obtained from rat cortex and SH-SY5Y human neuroblastoma cells, immunoblotting demonstrated enrichment of the 80kDa mu-calpain large subunit and 28kDa small subunit. In rat cortex, antibodies against domains II and III of the large mu-calpain subunit also detected a 40kDa fragment, similar to the autolytic fragment generated following incubation of human erythrocyte mu-calpain with Ca(2+). Mitochondrial proteins including apoptosis inducing factor and mitochondrial Bax are calpain substrates, but the mechanism by which calpains gain access to these proteins is uncertain. Mitochondrial localization of mu-calpain places the enzyme in proximity to its mitochondrial substrates and to Ca(2+) released from mitochondrial stores.     

Disruption of the mouse mu-calpain gene reveals an essential role in platelet function

Conventional calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. There are two forms of conventional calpains: the mu-calpain, or calpain I, which requires micromolar calcium for half-maximal activation, and the m-calpain, or calpain II, which functions at millimolar calcium concentrations. We evaluated the functional role of the 80-kDa catalytic subunit of mu-calpain by genetic inactivation using homologous recombination in embryonic stem cells. The mu-calpain-deficient mice are viable and fertile. The complete deficiency of mu-calpain causes significant reduction in platelet aggregation and clot retraction but surprisingly the mutant mice display normal bleeding times. No detectable differences were observed in the cleavage pattern and kinetics of calpain substrates such as the beta3 subunit of alphaIIbbeta3 integrin, talin, and ABP-280 (filamin). However, mu-calpain null platelets exhibit impaired tyrosine phosphorylation of several proteins including the beta3 subunit of alphaIIbbeta3 integrin, correlating with the agonist-induced reduction in platelet aggregation. These results provide the first direct evidence that mu-calpain is essential for normal platelet function, not by affecting the cleavage of cytoskeletal proteins but by potentially regulating the state of tyrosine phosphorylation of the platelet proteins.