Molecular dynamic simulations of the catalytic subunit of calpains 1, 2, 5, and 10: Structural analysis with an aim toward drug design

Calpains are cysteine proteases involved in the development of several human chronic illnesses such as neurodegenerative diseases, cardiovascular ailments, diabetes, and obesity which constitutes them into possible therapeutic targets. Here, using molecular dynamic simulations and docking, we studied the binding of known inhibitors to representative members of classical and nonclassical calpains. Our aim is to gain better understanding on the inhibition mechanism of calpains and to develop better and more specific inhibitors. Our atomistic models confirmed the importance of calcium ions for the structure of calpains and, as a consequence, their functionality. With these models and their subsequent use in molecular docking, essential structural requirements were identified for the binding of ligands to the calpain catalytic site that provide useful information for the design of new selective calpain inhibitors.     

The therapeutic potential of the calpain family: new aspects

The calpain family is a group of cysteine proteases unique in their dependency on calcium to attain functionally active forms. Calpains are involved in a wide range of cellular calcium-regulated functions, including signal transduction, cell proliferation and differentiation, and apoptosis. Moreover, altered calpain activity has been observed in several human diseases. Specific calpain inhibitors hold promise for the treatment of neuromuscular and neurodegenerative diseases in which calpains have been shown to be upregulated (e.g. Parkinson's disease and Duchenne muscular dystrophy). Conversely, calpain activators could be a useful approach for those diseases where reduced calpain activity has been observed, such as type 2 diabetes or metabolic syndrome.     

Calpains as potential anti-cancer targets

INTRODUCTION: The intracellular signaling cysteine proteases, calpains (specifically the ubiquitous calpains 1 and 2), are involved in numerous physiological and pathological phenomena. Several works have highlighted the implication of calpains in processes crucial for cancer development and progression. For these reasons, calpains are considered by several authors as potential anti-cancer targets. AREAS COVERED: How calpains are implicated in cancer formation and development, how these enzymes are deregulated in cancer cells and how these proteases could be targeted by anti-cancer drugs. Studies published in the last 10 years are focused on. EXPERT OPINION: Targeting calpain activity with specific inhibitors could be a novel approach to limiting development of primary tumors and formation of metastases, by inhibiting tumor cell migration and invasion, which allows dissemination as well as tumor neovascularization, which in turn allows expansion. However, such drugs could interfere with anti-cancer treatments, as ubiquitous calpains play crucial roles in chemotherapy-induced apoptosis. For these reasons, drugs targeting calpains would have to be used selectively to avoid interference with other treatments and physiological processes. Further studies will be required concerning the other members of the calpain family and their potential implication in cancer development before considering treatments targeting their activity.     

Developments in the design and synthesis of calpain inhibitors

Calpains are Ca(2+)-dependent cysteine proteases that play an important role in cell differentiation and in apoptosis/necrosis. The overactivation of calpain is connected with a number of diseases, including cataracts and traumatic brain injury, making calpain an attractive drug target. The development of selective inhibitors of calpain has, however, proved difficult, due to a lack of detailed structural information on the protein. This difficulty has been somewhat alleviated with recent reports on the X-ray crystal structures of engineered calpains and improved biochemical characterization of the protein. This review describes properties and X-ray crystal structures of calpain, and the synthesis and binding affinities of novel calpain inhibitors.     

Involvement of calpain activation in neurodegenerative processes

One of the challenges in the coming years will be to better understand the mechanisms of neuronal cell death with the objective of developing adequate drugs for the treatment of neurodegenerative disorders. Caspases and calpains are among the best-characterized cysteine proteases activated in brain disorders. Likewise, during the last decade, extensive research revealed that the deregulation of calpains activity is a key cytotoxic event in a variety of neurodegenerative disorders. Moreover, interest in the role of calpain in neurodegenerative processes is growing due to implication of the involvement of cdk5 in neurodegenerative diseases. Since calpain inhibitors appear to not only protect brain tissue from ischemia, but also to prevent neurotoxicity caused by such neurotoxins as beta-amyloid or 3-nitropropionic acid, the currently available data suggest that calpain and cdk5 play a key role in neuronal cell death. It seems clear that the inappropriate activation of cysteine proteases occurs not only during neuronal cell death, but may also contribute to brain pathology in ischemia and traumatic brain disorders. Pharmacological modulation of calpain activation may, therefore, be useful in the treatment of neurodegenerative disorders. It is possible, although difficult, to develop synthetic inhibitors of cysteine proteases, specifically calpains. The inhibition of calpain activation has recently emerged as a potential therapeutic target for the treatment of neurodegenerative diseases.     

Ritonavir inhibition of calcium-activated neutral proteases

Calpains (EC 3.4.22.17) are intracellular calcium-activated cysteine proteases that mediate tissue injury following post-ischemic and post-traumatic stress. Both human HIV protease and calpains share a similar secondary structure, where the active site is flanked by hydrophobic regions. The present study demonstrates that ritonavir, a hydrophobic HIV protease inhibitor, also inhibits calpain activity. In PC12 cell extracts assayed for calpain at maximal activity (2mM calcium), ritonavir exhibited competitive inhibition with a K(i) of 11+/-7.0 microM. Experiments with purified enzymes showed inhibition for both m- and mu-calpain isoforms (m-calpain, K(i)=9.2+/-1.2 microM; mu-calpain, K(i)=5.9+/-1.4 microM). Ritonavir also inhibited calcium-stimulated calpain activity in PC12 cells in situ. These results suggest that ritonavir or analogues of the drug should be investigated as cytoprotective agents in conditions where cell death or injury is mediated via calpain activation.     

Calpains as potential anti-cancer targets

Introduction: The intracellular signaling cysteine proteases, calpains (specifically the ubiquitous calpains 1 and 2), are involved in numerous physiological and pathological phenomena. Several works have highlighted the implication of calpains in processes crucial for cancer development and progression. For these reasons, calpains are considered by several authors as potential anti-cancer targets.

Areas covered: How calpains are implicated in cancer formation and development, how these enzymes are deregulated in cancer cells and how these proteases could be targeted by anti-cancer drugs. Studies published in the last 10 years are focused on.

Expert opinion: Targeting calpain activity with specific inhibitors could be a novel approach to limiting development of primary tumors and formation of metastases, by inhibiting tumor cell migration and invasion, which allows dissemination as well as tumor neovascularization, which in turn allows expansion. However, such drugs could interfere with anti-cancer treatments, as ubiquitous calpains play crucial roles in chemotherapy-induced apoptosis. For these reasons, drugs targeting calpains would have to be used selectively to avoid interference with other treatments and physiological processes. Further studies will be required concerning the other members of the calpain family and their potential implication in cancer development before considering treatments targeting their activity.     

Cocrystal structures of primed side-extending alpha-ketoamide inhibitors reveal novel calpain-inhibitor aromatic interactions

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 overactivation causes unregulated proteolysis, which can contribute to diseases such as postischemic 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 (muI-II) bound to two alpha-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 toward muI-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.     

Structure-activity relationship study and drug profile of N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (SJA6017) as a potent calpain inhibitor

Novel N-arylsulfonyldipeptidyl aldehyde derivatives were prepared by DMSO oxidation from the corresponding dipeptide alcohol, and their potencies as calpain inhibitors were evaluated in vitro. Among them, N-(4-fluorophenylsulfonyl)-l-valyl-l-leucinal (8, SJA6017) potently inhibited calpains. 8 also inhibited cathepsin B and L but did not inhibit other cysteine proteases (interleukin 1beta-converting enzyme), serine proteases (trypsin, chymotrypsin, thrombin, factor VIIa, factor Xa), or proteasome. Preliminary cytotoxicity studies of 8 exhibited a relatively safe profile.     

Calpains inhibitors--a review of the recent patent literature

Calpain inhibitors prevent proteolytic degradation of cellular proteins due to activation of calcium-activated cysteine proteases (calpains). As a class, they show promising cytoprotective activity in a variety of disorders where the final common pathway is increased intracellular concentrations of calcium, activation of calpain-mediated protein degradation, and subsequent cell death. These disorders include cerebral stroke, cerebral and spinal cord trauma, and myocardial infarction.     

钙蛋白酶与肝损伤

钙蛋白酶是一类依赖钙离子浓度激活的蛋白酶,通过剪切其底物蛋白发挥不可逆调节作用。此类蛋白酶广泛存在于人体各个脏器。钙蛋白酶参与细胞骨架和细胞膜的构型重建等重要生理过程。病理状态下钙蛋白酶的异常活化是脑外伤、脑缺血、脊髓损伤等神经元退行性变、白内障形成及缺血/再灌注损伤的重要机制之一。近年来钙蛋白酶在肝细胞损伤中的作用引起极大关注。该文将对钙蛋白酶基本生理功能及在肝损伤发展过程中的作用加以综述。     

Serine and Cysteine Proteases in Sulfur Mustard-Exposed Hairless Mouse Skin: Enzymatic Activity and Inhibition Profiles

Abstract

Inflammatory reactions associated with sulfur mustard (bis(2-chloroethyl)sulfide, SM)-induced skin pathology, including the release of potent proteolytic enzymes capable of destroying cutaneous connective tissue proteins, are not well defined in vivo. In this study, protease activities were measured as potential indicators of SM-induced skin injury in a hairless mouse sulfur mustard vapor exposure model. We also investigated the effects of synthetic protease inhibitors on SM-induced proteolytic activity. Skin homogenates prepared from hairless mouse dorsal skin exposed to SM vapor (1.4 g/m³) were assayed for serine and cysteine protease activities using synthetic chromogenic and fluorogenic substrates. Skin samples were obtained 24 h after SM challenge from animals exposed for either 6 or 12 min, while controls were obtained from unexposed skin. With 6 min SM-exposed skin, significantly higher elastase, tryptase, cathepsin B, cathepsin L, and calpain II enzymatic activities were detected compared to the unexposed control. With 12 min SM-exposed skin, significantly higher activities of elastase, tryptase, and calpain II were detected compared to the unexposed control. No significant differences were found between 6 and 12 min SM-induced protease activities, except 6-min cathepsin L, which was higher than the 12 min. Effective inhibitors include the general serine protease inhibitor 3,4-dichloroisocoumarin and specific peptide phosphonate serine protease inhibitors, in addition to peptide ketoamide transition-state cysteine protease inhibitors. These data demonstrate that proteases are involved in the process of SM-induced tissue injury, and suggest that the identification of specific inhibitors should be useful for studying cytotoxic mechanisms and reducing tissue injury caused by SM exposure.     

Currently evaluated calpain and caspase inhibitors for neuroprotection in experimental brain ischemia

Currently available therapies for brain ischemia, with a few exceptions, provide only symptomatic relief in patients. Recent investigations in experimental models provided an understanding of the cellular and molecular mechanisms that lead to neurodegeneration in ischemic injury, and also indicate targets for prevention and amelioration of the devastating consequences of stroke. An enormous increase in intracellular free Ca(2+) levels following stroke activates Ca(2+)-dependent enzymes, contributing to neuronal death and dysfunction. Additionally, ischemic injury generates highly reactive free radicals and triggers release of cytotoxic cytokines for activation of cysteine proteases. A number of studies already indicated a prominent role for the cysteine proteases of the calpain and caspase families in the pathogenesis of brain ischemia. Proteolytic activities of these proteases degrade various cytoskeletal proteins and membrane proteins, destabilizing the structural integrity and forcing the neurons to delayed death in ischemic penumbra. Some current studies have unequivocally confirmed the neuronal apoptosis in ischemia and showed that administration of calpain and caspase inhibitors alone or in combination can provide functional neuroprotection in various animal models of cerebral ischemia. This article will discuss the molecular structures and activities of calpain and caspase inhibitors and their therapeutic efficacy in experimental brain ischemia. However, further investigations are necessary for improvements in the structural design of calpain and caspase inhibitors for their persistent therapeutic efficacy in animal models of stroke and for clinical trials in the future.     

All cut up! The consequences of calpain activation on platelet function

The Ca(2+)-activated proteases or calpains, play a crucial role in a spectrum of physiological processes such as cytoskeletal remodeling, cellular signaling, cell migration, apoptosis, cell survival and platelet activation, by the proteolytic cleavage of target proteins. Rather than eliciting protein degradation the calpains are responsible for their modification (e.g. activation, inhibition or altered sensitivity to intracellular signals) and therefore make a significant impact on intracellular signaling. Maintained calpain activation is known to be associated with disease development and in platelet calpains are involved in both physiological platelet activation as well as pathological platelet hyper-activation.     

Cysteine cathepsin proteases as pharmacological targets in cancer

Proteolytic activity is required for several key pro-tumorigenic processes: angiogenesis, invasion and metastasis. Consequently, increases in protease expression and activity are frequently reported in human cancers, and correlate with malignant progression and poor patient prognosis. Cysteine cathepsin proteases have recently emerged as an important class of proteolytic enzymes in cancer development, and cysteine cathepsin inhibitors have been proposed as anticancer agents. In this review, we highlight recent studies that now allow us to evaluate critically whether cysteine cathepsin inhibition represents a viable therapeutic strategy for the treatment of cancer.     

Calpain and pathology in view of structure-function relationships

Calpain, a Ca(2+)-requiring cytoplasmic cysteine protease, plays indispensable roles in various cellular functions such as signal transduction, cell growth and differentiation, apoptosis, necrosis, and so on. Although most of the detailed physiological functions of calpains have not yet been elucidated, the importance of calpain is obvious from the increasing numbers of papers describing relationships between human disease states (such as Alzheimer's disease, cataract, and muscular dystrophies) and malfunction of calpain. One of the recent remarkable topics of calpain is that a single nucleotide polymorphism of CAPN10, the gene for calpain 10, is related to type 2 diabetes. However, physiological functions of calpain 10 and its relation to diabetes are still unclear. Among 14 human calpain genes, mutations in CAPN3, the gene for p94/calpain 3a and Lp82/calpain 3b, are the only example that genetically connects the calpain gene and human disease, in this case, limb-girdle muscular dystrophy type 2A (LGMD2A). p94 has unique characteristics such as apparent Ca(2+)-independent activation and very rapid autolytic activity, which are dependent on p94-specific regions, NS, IS1, and IS2. Based on the 3D structures of micro - and m-calpain, molecular functions of p94 in relation to LGMD2A are discussed, with the hope of providing us with some clues to understand calpain functions and its relationships to human diseases.     

Potential contribution of proteases to neuronal damage

Calpain was first discovered 30 years ago. Two major isoforms were subsequently isolated and purified. The presence of an endogenous protein inhibitor, calpastatin, was later discovered. Calpain activity is tightly regulated by Ca(2+). At physiological levels of Ca(2+), the role of calpain remains poorly understood, but is believed to be involved in mitosis and muscle cell differentiation. Calpain has also been implicated in various membrane fusion events through remodeling of the cytoskeletal network. Calpain activation has been shown to be increased during normal aging and in muscular dystrophy, cataract, arthritis and Alzheimer's disease, and in acute traumas such as traumatic brain injury (TBI), spinal cord injury and cerebral and cardiac ischemia. Early work on calpain inhibitors was limited to protein inhibitors and other nonselective enzyme inhibitors. Peptidyl aldehydes such as leupeptin and antipain are also among the earliest reported calpain inactivators. Irreversible inhibitors such as the E64 family have also been studied, and peptidyl halomethanes and diazomethanes have long been used as protease inhibitors. A variety of calpain inhibitors are under development. From a therapeutic perspective, calpain inhibitors may have several advantages over other more conventional targets such as ion channel blockers and glumate antagonists, since calpain proteolysis represents a later component of a pathway mediating cell death initiated by excitotoxicity and elevated Ca(2+) levels. Although the potential clinical utility of calpain inhibitors seems well established, a number of important considerations remain to be addressed. The role of other proteolytic cascades contributing to neuronal cell damage following TBI must also be considered.     

Role of calpain and caspase in beta-amyloid-induced cell death in rat primary septal cultured neurons

The invariant characteristic features associated with Alzheimer's disease (AD) brain include the presence of extracellular neuritic plaques composed of amyloid beta (Abeta) peptide, intracellular neurofibrillary tangles containing hyper-phosphorylated tau protein and the loss of basal forebrain cholinergic neurons. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that in vivo accumulation of Abeta(1-42) may initiate the process of neurodegeneration observed in AD brains. However, the cause of degeneration of the basal forebrain cholinergic neurons and their association to Abeta peptides or phosphorylated tau protein have not been clearly established. In the present study, using rat primary septal cultures, we have shown that Abeta(1-42), in a time (1-48 h) and concentration (0.01-20 microM)-dependent manner, induce toxicity in cultured neurons. Subsequently, we have demonstrated that Abeta toxicity is mediated via activation of cysteine proteases, i.e., calpain and caspase, and proteolytic breakdown of their downstream substrates tau, microtubule-associated protein-2 and alpha II-spectrin. Additionally, Abeta-treatment was found to induce phosphorylation of tau protein along with decreased levels of phospho-Akt and phospho-Ser(9)glycogen synthase kinase-3beta. Exposure to specific inhibitors of caspase or calpain can partially protect cultured neurons against Abeta-induced toxicity but their effects are not found to be additive. These results, taken together, suggest that Abeta peptide can induce toxicity in rat septal cultured neurons by activating multiple intracellular signaling molecules. Additionally, evidence that inhibitors of caspase and calpains can partially protect the cultured basal forebrain neurons raised the possibility that their inhibitors could be of therapeutic relevance in the treatment of AD pathology.     

New peptidic cysteine protease inhibitors derived from the electrophilic alpha-amino acid aziridine-2,3-dicarboxylic acid

Three different types of peptides containing aziridine-2, 3-dicarboxylic acid (Azi) as an electrophilic alpha-amino acid at different positions within the peptide chain (type I, N-acylated aziridines with Azi as C-terminal amino acid; type II, N-unsubstituted aziridines with Azi as N-terminal amino acid; type III, N-acylated bispeptidyl derivatives of Azi) have been synthesized and tested as inhibitors of the cysteine proteases papain, cathepsins B, L, and H, and calpains I and II, as well as against several serine proteases, one aspartate, and one metalloprotease. All aziridinyl peptides are specific cysteine protease inhibitors. Papain and cathepsins B and L are inhibited irreversibly, whereas cathepsin H and calpains are inhibited in a non-time-dependent manner. Some compounds turned out to be substrates for serine proteases and for the metalloprotease thermolysin. Remarkable differences can be observed between the three different types of inhibitors concerning stereospecificity, pH dependency of inhibition, selectivity between different cysteine proteases, and the importance of a free carboxylic acid function at the aziridine ring for inhibition. Above all type II inhibitors, aza analogues of the well-known epoxysuccinyl peptides, are potent cysteine protease inhibitors. With the exception of BOC-Leu-Gly-(S, S+R,R)-Azi-(OEt)2 (28a+b), a highly selective and potent cathepsin L inhibitor, N-acylated aziridines of type I are weaker inhibitors than type II or type III compounds. The observed results can be explained by different binding modes of the three types of inhibitors with respect to their orientation in the S- and S'-binding sites of the enzymes. Furthermore, the presence of a protonated aziridine N modifies the binding mode of type II inhibitors.     

Michael acceptors as cysteine protease inhibitors

Cysteine proteases selectively catalyze the hydrolysis of peptide bonds. Uncontrolled, unregulated, or undesired proteolysis can lead to many disease states including emphysema, stroke, viral infections, cancer, Alzheimer's disease, inflammation, and arthritis. Cysteine proteases inhibitors thus have considerable potential utility for therapeutic intervention in a variety of disease states. This review emphasizes on the new developments from literature reports on Michael acceptors as potential cysteine protease inhibitors, namely vinyl sulfones, alpha,beta-unsaturated carbonyl derivatives and aza-peptides. These compounds irreversibly alkylate the active site cysteine residue via conjugate addition. Examples of Michael acceptors inhibitors that have already progressed to clinical testing are also presented.